by the Autophagic/Lysosomal and Proteasomal
Pathways in Atrophying Muscle Cells
Jinghui Zhao,1Jeffrey J. Brault,1Andreas Schild,1Peirang Cao,2Marco Sandri,3,4,5Stefano Schiaffino,3,4,6
Stewart H. Lecker,2and Alfred L. Goldberg1,*
1Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA
2Renal Unit, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02115, USA
3Venetian Institute of Molecular Medicine, 35129 Padova, Italy
4Department of Biomedical Sciences, University of Padova, 35121 Padova, Italy
5Dulbecco Telethon Institute, 35129 Padova, Italy
6Institute of Neuroscience, Consiglio Nazionale delle Ricerche, 35121 Padova, Italy
Muscle atrophy occurs in many pathological
states and results primarily from accelerated
protein degradation and activation of the ubiq-
uitin-proteasome pathway. However, the im-
portance of lysosomes in muscle atrophy has
scription factors is essential for the atrophy in-
duced by denervation or fasting, and activated
FoxO3 by itself causes marked atrophy of mus-
cles and myotubes. Here, we report that FoxO3
does so by stimulating overall protein degrada-
tion and coordinately activating both lysosomal
and proteasomal pathways. Surprisingly, in
C2C12 myotubes, most of this increased pro-
teolysis is mediated by lysosomes. Activated
cle (and other cell types) by activating autoph-
agy. FoxO3 also induces the expression of
many autophagy-related genes, which are in-
duced similarly in mouse muscles atrophying
due to denervation or fasting. These studies in-
dicate that decreased IGF-1-PI3K-Akt signaling
activates autophagy not only through mTOR
but also more slowly by a transcription-depen-
dent mechanism involving FoxO3.
Muscle atrophyisadebilitating processthatleadstorapid
loss of strength and endurance. It occurs in specific mus-
cles with inactivity and denervation and systematically in
fasting and many diseases, including cancer, diabetes,
sepsis, and renal failure (Kandarian and Jackman, 2006;
Lecker et al., 2006). In these various conditions, the rapid
loss of muscle mass occurs primarily through an activa-
tion of protein breakdown. We have previously identified
a set of atrophy-specific genes or ‘‘atrogenes’’ that are
up- or downregulated similarly in muscles in these diverse
These observations indicate that common transcriptional
adaptations occur in various types of atrophy leading to
accelerated protein degradation.
Proteasomes and lysosomes comprise the two major
intracellular proteolytic systems in mammalian cells and
have generally been assumed to be regulated indepen-
dently and to serve distinct functions. The ubiquitin-pro-
teasome pathway degrades both cytosolic and nuclear
proteins (Glickman and Ciechanover, 2002), as well as
comprise most of the protein in adult skeletal muscle. The
acid hydrolases in lysosomes degrade most membrane
and extracellular proteins taken up by endocytosis, as
well as cytoplasmic proteins and organelles through au-
tophagy (Scott and Klionsky, 1998). In diverse types of
muscle wasting, the ubiquitin-proteasome pathway is ac-
tivated, as shown by increased sensitivity to proteasome
inhibitors; increased levels of ubiquitin conjugates; en-
hanced rates of ubiquitin conjugation; and induction of
genes for ubiquitin, several proteasomal subunits, and
two critical ubiquitin ligases (E3s), atrogin-1/MAFbx and
MuRF1 (Bodine et al., 2001; Lecker et al., 2004). Induction
of these muscle-specific E3s is essential for rapid atrophy
(Bodineetal., 2001). Because inhibitorsof lysosomalfunc-
in atrophying muscles (Furuno et al., 1990), the possible
contributions of lysosomes to atrophy have not attracted
much attention. However, an increased capacity for lyso-
somal proteolysis has been demonstrated in various types
of atrophy (Bechet et al., 2005; Furuno et al., 1990).
Our prior work showed that activation of FoxO tran-
scription factors is essential for fiber atrophy and atro-
gin-1 induction upon denervation, fasting, and glucocorti-
coid treatment (Sandri et al., 2004, 2006). Moreover,
expression of constitutively active FoxO3 (ca-FoxO3)
induces expression of multiple atrogenes, including the
critical E3 atrogin-1, and causes dramatic atrophy of
mouse muscles and myotubes (Sandri et al., 2004). The
472 Cell Metabolism 6, 472–483, December 2007 ª2007 Elsevier Inc.
to their inactivation through binding to 14-3-3 proteins in
the cytosol (Tran et al., 2003), but in ca-FoxO3, the three
Akt phosphorylation sites are mutated to alanines, allow-
ing free entry into the nucleus. Through activation of the
PI3K-Akt pathway, IGF-1 stimulates protein synthesis
and can induce hypertrophy of skeletal muscle (Glass,
2005). In addition, IGF-1/insulin inhibits overall protein
breakdown, degradation of myofibrillar proteins (Sacheck
et al., 2004), and expression of atrogin-1 and MuRF1
(Sacheck et al., 2004; Stitt et al., 2004). This reduced pro-
teolysis appears to contribute to muscle growth and to re-
sult from the inactivation of FoxO by Akt phosphorylation
(Sacheck et al., 2004; Stitt et al., 2004), while the acceler-
Sandri et al., 2004). The present studies were undertaken
to analyze how FoxO3 affects protein degradation and to
assess the contributions of lysosomal and proteasomal
pathways to the loss of muscle protein during atrophy.
We demonstrate here a marked ability of FoxO3 to stimu-
late lysosomal proteolysis by activating autophagy via
a transcriptional mechanism in muscle cells (as well as
is activated coordinately with the proteasomal pathway
and contributes importantly to atrophy.
FoxO3 Causes Atrophy by Stimulating Proteolysis
To determine the effects of FoxO3 on protein degrada-
tion, we infected C2C12 myotubes with an adenovirus
expressing ca-FoxO3, which within 2 days caused a dra-
matic reduction in myotube diameter (Figure 1A) and
a reduction in the protein:DNA ratio (data not shown) to
a level below that observed in control cells infected
with an adenovirus expressing GFP only. Thus, viral ex-
pression of ca-FoxO3 in myotubes mimics the effects
of FoxO3 overexpression in adult muscle (Sandri et al.,
To measure overall rates of protein degradation, the
majority of the myotube proteins were labeled by a 20 hr
exposure to [3H]tyrosine prior to viral infection. After infec-
tion, overall rates of protein degradation were determined
by measuring [3H]tyrosine release from the prelabeled
proteins into the medium, which contained a large excess
of nonradioactive tyrosine to prevent reincorporation
(Gronostajski et al., 1984). After 16 hr exposure to the
ca-FoxO3-expressing adenovirus, the overall rate of pro-
tein degradation in the myotubes was significantly in-
creased (Figure 1B, p < 0.01), and by 24 hr, the rate was
at least 50% greater than in cells infected with control
adenovirus (Figure 1B and Figure 2A). If maintained, this
enhancement of proteolysis is sufficient to cause marked
atrophy in 2 days. When expressed at a level similar to
of proteolysis by only 15% (Figure 1B, p < 0.01), presum-
ably because most of the wt-FoxO3 was phosphorylated
FoxO3-Induced Increase in Proteolysis
in Myotubes Is Mediated Mainly by Lysosomes
To determine to what extent proteasomes contribute to
FoxO3-induced proteolysis, we used two specific protea-
some inhibitors, Velcade (PS-341) and lactacystin (Kisse-
lev and Goldberg, 2001). To evaluate the contributions of
lysosomes, we used a specific inhibitor of the lysosomal
proton pump, concanamycin A (Woo et al., 1992), or chlo-
roquine and NH4Cl, which accumulate in lysosomes and
raise intralysosomal pH (Seglen, 1983). At concentrations
used, the inhibitors caused maximal inhibition of proteoly-
sis, and residual rates of degradation were linear for up to
5 hr with no detectable cell death. The actual contribution
of proteasomes or lysosomes was then determined as the
somal inhibitors, by subtracting the rates of proteolysis in
Figure 1. FoxO3 Induces Atrophy and
(A) Constitutively active FoxO3 (ca-FoxO3) in-
duces marked atrophy. Myotubes were infected
with control (top panel, expressing only GFP) or
ca-FoxO3 (bottom panel, expressing ca-FoxO3
and GFP) adenoviruses. Cells expressing GFP
are shown 48 hr after infection.
(B) ca-FoxO3 stimulates proteolysis. Myotubes
were incubated with [3H]tyrosine for 20 hr and
then washed with chase medium for 2 hr. New
chase media containing control, ca-FoxO3, or
were added, and media samples were collected
over 36 hr. The released radioactivity (indicating
proteins degraded) was plotted as a percentage
of total [3H]tyrosine incorporated into cell pro-
teins. The rates of proteolysis (calculated from
the linear slopes between 20 and 32 hr) are
also shown. Levels of FoxO3 protein at different
times after infection were analyzed by western
blot. Error bars represent SEM.
FoxO3 Stimulates Autophagy in Atrophying Muscle
Cell Metabolism 6, 472–483, December 2007 ª2007 Elsevier Inc. 473
EDTA, 0.1 mM Na3VO4, and protease inhibitor cocktail [Roche]). Thirty
micrograms of total proteins was separated by SDS-PAGE, trans-
ferred to PVDF membranes, and analyzed by western blot using the
ECL method (Amersham). We used polyclonal antibodies against
FoxO3, p-FoxO3(Thr32) (Upstate), 4E-BP1, S6K, p-S6K(Thr389),
mTOR, p-mTOR(Ser2448) (Cell Signaling), b-actin (Abcam), LC3, be-
clin1 (Santa Cruz), Gabarapl1 (Proteintech Group, Inc.). Protein levels
were quantified by using Quantity One software.
Chromatin Immunoprecipitation Assay
Myotubes were infected with control or ca-FoxO3 adenoviruses for
12 hr before analysis using the chromatin immunoprecipitation (ChIP)
assay kit (Upstate). Cellular chromatin was crosslinked by formalde-
hyde and then sheared by sonication. The soluble chromatin was im-
munoprecipitated with an anti-HA polyclonal antibody (Santa Cruz).
After decrosslinking, the immunoprecipitates were subjected to
PCR. Primers used are listed in Table S2.
Generation of atrogin-1-Deficient Mice and Primary Muscle
atrogin-1knockoutmice(Bodine etal.,2001)wereprovided byRegen-
eron. Myoblasts were isolated using the procedure of Rando and Blau
(1994).Muscles were removed from the hindlimbs of 2-week-old mice.
After treatment with 0.1% collagenase D and dispase II (Roche), the
isolated cells were plated on collagen-coated dishes. Myoblasts
were cultured in F-10 nutrient medium with 20% fetal calf serum,
2.5 ng/ml basic fibroblast growth factor (Invitrogen). Myotubes
were induced by switching to differentiation medium. All media
contained Primocin (InvivoGen). Cultures were used on the third day
in differentiation medium, when myotubes formed.
Single-Fiber Analyses of Adult Mouse Muscle
Flexor digitorum brevis muscles from adult mice were digested in type
I collagenase at 4?C for 1 hr, at 37?C for 2 hr, and dissociated into
single fibers. The fibers were electroporated using a BTX porator
(50 volts/4 mm, 3 pulses, 200 ms intervals) to transfer plasmid DNA
and then plated on glass coverslips coated with laminin and cultured
in Tyrode’s salt solution (pH 7.3) containing 10% fetal bovine serum,
50 U/ml penicillin, 50 mg/ml streptomycin, and 5% CO2(37?C). The
shRNA construct used for knocking down FoxO3 was described pre-
viously (Sandri et al., 2004). The specific sequence used against
FoxO3 was 50-GGATAAGGGCGACAGCAAC-30.
with this article online at http://www.cellmetabolism.org/cgi/content/
This work was supported by grants to A.L.G. from the Muscular Dys-
trophy Association, the National Space Biomedical Research Institute,
the Fund for Innovation from Elan Pharmaceuticals, and the Ellison
Foundation. J.J.B. was supported by a Ruth L. Kirschstein National
ship from the Swiss National Science Foundation. M.S. wassupported
by grants from the Agenzia Spaziale Italiana (OSMA project), Telethon
Italy (TCP04009), and Compagnia San Paolo. S.S. was supported by
grants from the Agenzia Spaziale Italiana (OSMA project) and the EU
2004-005272). S.H.L. was supported by grant DK062307 from the
NIH. We thank Y. Ohsumi for providing Atg5-deficient MEFs and
A.M. Cuervo for providing Lamp2a primers.
Received: July 10, 2007
Revised: September 30, 2007
Accepted: November 6, 2007
Published: December 4, 2007
Arico, S., Petiot, A., Bauvy, C., Dubbelhuis, P.F., Meijer, A.J.,
Codogno, P., and Ogier-Denis, E. (2001). The tumor suppressor
PTEN positively regulates macroautophagy by inhibiting the phospha-
tidylinositol 3-kinase/protein kinase B pathway. J. Biol. Chem. 276,
Barthel, A., Schmoll, D., and Unterman, T.G. (2005). FoxO proteins
in insulin action and metabolism. Trends Endocrinol. Metab. 16,
Bechet, D., Tassa, A., Taillandier, D., Combaret, L., and Attaix, D.
(2005). Lysosomal proteolysis in skeletal muscle. Int. J. Biochem.
Cell Biol. 37, 2098–2114.
Beehler, B.C., Sleph, P.G., Benmassaoud, L., and Grover, G.J. (2006).
Reduction of skeletal muscle atrophy by a proteasome inhibitor in a rat
model of denervation. Exp. Biol. Med. (Maywood) 231, 335–341.
Bodine, S.C., Latres, E., Baumhueter, S., Lai, V.K., Nunez, L., Clarke,
B.A., Poueymirou, W.T., Panaro, F.J., Na, E., Dharmarajan, K., et al.
(2001). Identification of ubiquitin ligases required for skeletal muscle
atrophy. Science 294, 1704–1708.
Brunet, A., Bonni, A., Zigmond, M.J., Lin, M.Z., Juo, P., Hu, L.S.,
Anderson, M.J., Arden, K.C., Blenis, J., and Greenberg, M.E. (1999).
Akt promotes cell survival by phosphorylating and inhibiting
a Forkhead transcription factor. Cell 96, 857–868.
Cuervo, A.M., and Dice, J.F. (2000). Unique properties of lamp2a
compared to other lamp2 isoforms. J. Cell Sci. 113, 4441–4450.
Dice, J.F. (2007). Chaperone-mediated autophagy. Autophagy 3, 295–
Du, J., Wang, X., Miereles, C., Bailey, J.L., Debigare, R., Zheng, B.,
Price, S.R., and Mitch, W.E. (2004). Activation of caspase-3 is an initial
step triggering accelerated muscle proteolysis in catabolic conditions.
J. Clin. Invest. 113, 115–123.
Furuno, K., Goodman, M.N., and Goldberg, A.L. (1990). Role of differ-
ent proteolytic systems in the degradation of muscle proteins during
denervation atrophy. J. Biol. Chem. 265, 8550–8557.
Glass, D.J. (2005). Skeletal muscle hypertrophy and atrophy signaling
pathways. Int. J. Biochem. Cell Biol. 37, 1974–1984.
Glickman, M.H., and Ciechanover, A. (2002). The ubiquitin-protea-
some proteolytic pathway: destruction for the sake of construction.
Physiol. Rev. 82, 373–428.
Gronostajski, R.M., Goldberg, A.L., and Pardee, A.B. (1984). The role
of increased proteolysis in the atrophy and arrest of proliferation in
serum-deprived fibroblasts. J. Cell. Physiol. 121, 189–198.
Iwata, A., Riley, B.E., Johnston, J.A., and Kopito, R.R. (2005). HDAC6
and microtubules are required for autophagic degradation of aggre-
gated huntingtin. J. Biol. Chem. 280, 40282–40292.
Juhasz, G., Puskas, L.G., Komonyi, O., Erdi, B., Maroy, P., Neufeld,
T.P., and Sass, M. (2007). Gene expression profiling identifies
FKBP39 as an inhibitor of autophagy in larval Drosophila fat body.
Cell Death Differ. 14, 1181–1190.
Kandarian, S.C., and Jackman, R.W. (2006). Intracellular signaling
during skeletal muscle atrophy. Muscle Nerve 33, 155–165.
Kisselev, A.F., and Goldberg, A.L. (2001). Proteasome inhibitors: from
research tools to drug candidates. Chem. Biol. 8, 739–758.
Klionsky, D.J., Cregg, J.M., Dunn, W.A., Jr., Emr, S.D., Sakai, Y.,
Sandoval, I.V., Sibirny, A., Subramani, S., Thumm, M., Veenhuis, M.,
and Ohsumi, Y. (2003). A unified nomenclature for yeast autophagy-
related genes. Dev. Cell 5, 539–545.
Knecht, E., Hernandez-Yago, J., and Grisolia, S. (1984). Regulation of
lysosomal autophagy in transformed and non-transformed mouse
fibroblasts under several growth conditions. Exp. Cell Res. 154, 224–
Kramerova, I., Kudryashova, E., Venkatraman, G., and Spencer, M.J.
(2005). Calpain 3 participates in sarcomere remodeling by acting
482 Cell Metabolism 6, 472–483, December 2007 ª2007 Elsevier Inc.
FoxO3 Stimulates Autophagy in Atrophying Muscle
upstream of the ubiquitin-proteasome pathway. Hum. Mol. Genet. 14,
Lecker, S.H., Jagoe, R.T., Gilbert, A., Gomes, M., Baracos, V., Bailey,
skeletal muscle atrophy involve a common program of changes in
gene expression. FASEB J. 18, 39–51.
Lecker, S.H., Goldberg, A.L., and Mitch, W.E. (2006). Protein degrada-
tion by the ubiquitin-proteasome pathway in normal and disease
states. J. Am. Soc. Nephrol. 17, 1807–1819.
Levine, B., and Klionsky, D.J. (2004). Development by self-digestion:
molecular mechanisms and biological functions of autophagy. Dev.
Cell 6, 463–477.
Liang, X.H., Jackson, S., Seaman, M., Brown, K., Kempkes, B.,
Hibshoosh, H., and Levine, B. (1999). Induction of autophagy and inhi-
bition of tumorigenesis by beclin 1. Nature 402, 672–676.
Mammucari, C., Milan, G., Romanello, V., Masiero, E., Rudolf, R., Del
Piccolo, P., Burden, S.J., Di Lisi, R., Sandri, C., Zhao, J., et al. (2007).
Meijer,A.J., and Codogno, P. (2006). Signalling and autophagy regula-
tion in health, aging and disease. Mol. Aspects Med. 27, 411–425.
Melendez, A.,Talloczy, Z., Seaman, M.,Eskelinen,E.L., Hall, D.H., and
Levine, B. (2003). Autophagy genes are essential for dauer develop-
ment and life-span extension in C. elegans. Science 301, 1387–1391.
Mizushima, N., Yamamoto, A., Hatano, M., Kobayashi, Y., Kabeya, Y.,
Suzuki, K., Tokuhisa, T., Ohsumi, Y., and Yoshimori, T. (2001). Dissec-
tion of autophagosome formation using Apg5-deficient mouse embry-
onic stem cells. J. Cell Biol. 152, 657–668.
Mizushima, N., Yamamoto, A., Matsui, M., Yoshimori, T., and Ohsumi,
Y. (2004). In vivo analysis of autophagy in response to nutrient starva-
tion using transgenic mice expressing a fluorescent autophagosome
marker. Mol. Biol. Cell 15, 1101–1111.
Mortimore, G.E., Hutson, N.J., and Surmacz, C.A. (1983). Quantitative
correlation between proteolysis and macro- and microautophagy in
mouse hepatocytes during starvation and refeeding. Proc. Natl.
Acad. Sci. USA 80, 2179–2183.
Ohsumi, Y. (2001). Molecular dissection of autophagy: two ubiquitin-
like systems. Nat. Rev. Mol. Cell Biol. 2, 211–216.
Petiot, A., Ogier-Denis, E., Blommaart, E.F., Meijer, A.J., and
Codogno,P.(2000). Distinctclassesof phosphatidylinositol30-kinases
are involved in signaling pathways that control macroautophagy in
HT-29 cells. J. Biol. Chem. 275, 992–998.
Rando, T.A., and Blau, H.M. (1994). Primary mouse myoblast purifica-
tion, characterization, and transplantation for cell-mediated gene ther-
apy. J. Cell Biol. 125, 1275–1287.
Rock, K.L., Gramm, C., Rothstein, L., Clark, K., Stein, R., Dick, L.,
Hwang, D., and Goldberg, A.L. (1994). Inhibitors of the proteasome
block the degradation of most cell proteins and the generation of pep-
tides presented on MHC class I molecules. Cell 78, 761–771.
Sacheck, J.M., Ohtsuka, A., McLary, S.C., and Goldberg, A.L. (2004).
IGF-I stimulates muscle growth by suppressing protein breakdown
and expression of atrophy-related ubiquitin ligases, atrogin-1 and
MuRF1. Am. J. Physiol. Endocrinol. Metab. 287, E591–E601.
Sacheck, J.M., Hyatt, J.P., Raffaello, A., Jagoe, R.T., Roy, R.R.,
Edgerton, V.R., Lecker, S.H., and Goldberg, A.L. (2007). Rapid disuse
and denervation atrophy involve transcriptional changes similar to
those of muscle wasting during systemic diseases. FASEB J. 21,
Sandri, M., Sandri, C., Gilbert, A., Skurk, C., Calabria, E., Picard, A.,
Walsh, K., Schiaffino, S., Lecker, S.H., and Goldberg, A.L. (2004).
Foxo transcription factors induce the atrophy-related ubiquitin ligase
atrogin-1 and cause skeletal muscle atrophy. Cell 117, 399–412.
Sandri, M., Lin, J., Handschin, C., Yang, W., Arany, Z.P., Lecker, S.H.,
Goldberg, A.L., and Spiegelman, B.M. (2006). PGC-1alpha protects
skeletal muscle from atrophy by suppressing FoxO3 action and atro-
phy-specific gene transcription. Proc. Natl. Acad. Sci. USA 103,
Sarkar, S., Davies, J.E., Huang, Z., Tunnacliffe, A., and Rubinsztein,
D.C. (2007). Trehalose, a novel mTOR-independent autophagy
enhancer, accelerates the clearance of mutant huntingtin and alpha-
synuclein. J. Biol. Chem. 282, 5641–5652.
Schiaffino, S., and Hanzlikova, V. (1972). Studies on the effect of
denervation in developing muscle. II. The lysosomal system. J. Ultra-
struct. Res. 39, 1–14.
Scott, S.V., and Klionsky, D.J. (1998). Delivery of proteins and organ-
elles to the vacuole from the cytoplasm. Curr. Opin. Cell Biol. 10,
Seglen, P.O. (1983). Inhibitors of lysosomal function. Methods
Enzymol. 96, 737–764.
Seglen, P.O., and Gordon, P.B. (1982). 3-Methyladenine: specific
inhibitor of autophagic/lysosomal protein degradation in isolated rat
hepatocytes. Proc. Natl. Acad. Sci. USA 79, 1889–1892.
Solomon, V., and Goldberg, A.L. (1996). Importance of the ATP-ubiq-
uitin-proteasome pathway in the degradation of soluble and myofibril-
lar proteins in rabbit muscle extracts. J. Biol. Chem. 271, 26690–
Stitt, T.N., Drujan, D., Clarke, B.A., Panaro, F., Timofeyva, Y., Kline,
W.O., Gonzalez, M., Yancopoulos, G.D., and Glass, D.J. (2004). The
IGF-1/PI3K/Akt pathway prevents expression of muscle atrophy-in-
duced ubiquitin ligases by inhibiting FOXO transcription factors. Mol.
Cell 14, 395–403.
Tassa, A., Roux, M.P., Attaix, D., and Bechet, D.M. (2003). Class III
phosphoinositide 3-kinase-Beclin1 complex mediates the amino
acid-dependent regulation of autophagy in C2C12 myotubes.
Biochem. J. 376, 577–586.
Tran, H., Brunet, A., Griffith, E.C., and Greenberg, M.E. (2003). The
many forks in FOXO’s road. Sci. STKE 2003, RE5.
Woo, J.T., Shinohara, C., Sakai, K., Hasumi, K., and Endo, A. (1992).
Isolation, characterization and biological activities of concanamycins
as inhibitors of lysosomal acidification. J. Antibiot. (Tokyo) 45, 1108–
Yamamoto, A., Cremona, M.L., and Rothman, J.E. (2006). Autophagy-
mediated clearance of huntingtin aggregates triggered by the insulin-
signaling pathway. J. Cell Biol. 172, 719–731.
Yang, L., Dan, H.C., Sun, M., Liu, Q., Sun, X.M., Feldman, R.I.,
Hamilton, A.D., Polokoff, M., Nicosia, S.V., Herlyn, M., et al. (2004).
Akt/protein kinase B signaling inhibitor-2, a selective small molecule
inhibitor of Akt signaling with antitumor activity in cancer cells overex-
pressing Akt. Cancer Res. 64, 4394–4399.
Cell Metabolism 6, 472–483, December 2007 ª2007 Elsevier Inc. 483
FoxO3 Stimulates Autophagy in Atrophying Muscle