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Insulin-like Growth Factor-1 (IGF-1) Inversely Regulates Atrophy-induced Genes via the Phosphatidylinositol 3-Kinase/Akt/Mammalian Target of Rapamycin (PI3K/Akt/mTOR) Pathway*

Open AccessPublished:November 17, 2004DOI:https://doi.org/10.1074/jbc.M407517200
      Skeletal muscle size is regulated by anabolic (hypertrophic) and catabolic (atrophic) processes. We first characterized molecular markers of both hypertrophy and atrophy and identified a small subset of genes that are inversely regulated in these two settings (e.g. up-regulated by an inducer of hypertrophy, insulin-like growth factor-1 (IGF-1), and down-regulated by a mediator of atrophy, dexamethasone). The genes identified as being inversely regulated by atrophy, as opposed to hypertrophy, include the E3 ubiquitin ligase MAFbx (also known as atrogin-1). We next sought to investigate the mechanism by which IGF-1 inversely regulates these markers, and found that the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway, which we had previously characterized as being critical for hypertrophy, is also required to be active in order for IGF-1-mediated transcriptional changes to occur. We had recently demonstrated that the IGF1/PI3K/Akt pathway can block dexamethasone-induced up-regulation of the atrophy-induced ubiquitin ligases MuRF1 and MAFbx by blocking nuclear translocation of a FOXO transcription factor. In the current study we demonstrate that an additional step of IGF1 transcriptional regulation occurs downstream of mTOR, which is independent of FOXO. Thus both the Akt/FOXO and the Akt/mTOR pathways are required for the transcriptional changes induced by IGF-1.
      Skeletal muscle mass and fiber size is regulated in response to changes in workload, activity, conditions such as AIDS, cancer, and aging, and by cachectic glucocorticoids such as dexamethasone (
      • Glass D.J.
      ,
      • Glass D.J.
      ,
      • Jagoe R.T.
      • Goldberg A.L.
      ). An increase in adult muscle mass and fiber size is called “hypertrophy” and is associated with increased protein synthesis (
      • Goldspink D.F.
      • Garlick P.J.
      • McNurlan M.A.
      ). A decrease in mass, called “atrophy,” is characterized by enhanced protein degradation (
      • Jagoe R.T.
      • Goldberg A.L.
      ,
      • Hasselgren P.O.
      ,
      • Mitch W.E.
      • Goldberg A.L.
      ).
      Hypertrophy in adult skeletal muscle is accompanied by the increased expression of insulin-like growth factor-1 (IGF-1)
      The abbreviations used are: IGF-1, insulin-like growth factor-1; PI3K, phosphatidylinositol 3-kinase; mTOR, mammalian target of rapamycin; DEX, dexamethasone; D2 and D3, day 2 and day 3, respectively; RAP, rapamycin; MT, metallothionein; PLF, proliferin; PV, parvalbumin; LY, LY294002.
      1The abbreviations used are: IGF-1, insulin-like growth factor-1; PI3K, phosphatidylinositol 3-kinase; mTOR, mammalian target of rapamycin; DEX, dexamethasone; D2 and D3, day 2 and day 3, respectively; RAP, rapamycin; MT, metallothionein; PLF, proliferin; PV, parvalbumin; LY, LY294002.
      (
      • Goldspink D.F.
      • Garlick P.J.
      • McNurlan M.A.
      ,
      • DeVol D.L.
      • Rotwein P.
      • Sadow J.L.
      • Novakofski J.
      • Bechtel P.J.
      ). When IGF-1 was overexpressed in the skeletal muscle of transgenic mice an increase in muscle size resulted (
      • Coleman M.E.
      • DeMayo F.
      • Yin K.C.
      • Lee H.M.
      • Geske R.
      • Montgomery C.
      • Schwartz R.J.
      ,
      • Musaro A.
      • McCullagh K.
      • Paul A.
      • Houghton L.
      • Dobrowolny G.
      • Molinaro M.
      • Barton E.R.
      • Sweeney H.L.
      • Rosenthal N.
      ). Furthermore, addition of IGF-1 in vitro to differentiated muscle cells promotes myotube hypertrophy (
      • Florini J.R.
      • Ewton D.Z.
      • Coolican S.A.
      ,
      • Rommel C.
      • Clarke B.A.
      • Zimmermann S.
      • Nunez L.
      • Rossman R.
      • Reid K.
      • Moelling K.
      • Yancopoulos G.D.
      • Glass D.J.
      ,
      • Rommel C.
      • Bodine S.C.
      • Clarke B.A.
      • Rossman R.
      • Nunez L.
      • Stitt T.N.
      • Yancopoulos G.D.
      • Glass D.J.
      ), supporting the idea that IGF-1 is sufficient to induce hypertrophy. The binding of IGF-1 to its receptor triggers the activation of phosphatidylinositol 3-kinase (PI3K). PI3K phosphorylates the membrane phospholipid phosphatidylinositol 4,5-bisphosphate to produce phosphatidylinositol 3,4,5-trisphosphate (
      • Matsui T.
      • Nagoshi T.
      • Rosenzweig A.
      ,
      • Vivanco I.
      • Sawyers C.L.
      ), creating a lipid binding site on the cell membrane for the serine/threonine kinase Akt (also called Akt1 and PKB, for protein kinase B) (
      • Alessi D.R.
      • James S.R.
      • Downes C.P.
      • Holmes A.B.
      • Gaffney P.R.
      • Reese C.B.
      • Cohen P.
      ,
      • Andjelkovic M.
      • Alessi D.R.
      • Meier R.
      • Fernandez A.
      • Lamb N.J.
      • Frech M.
      • Cron P.
      • Cohen P.
      • Lucocq J.M.
      • Hemmings B.A.
      ,
      • Andjelkovic M.
      • Jakubowicz T.
      • Cron P.
      • Ming X-F.
      • Han J.-W.
      • Hemmings B.A.
      ). The subsequent translocation of Akt to the membrane facilitates its phosphorylation and activation by the kinase PDK-1 (
      • Vivanco I.
      • Sawyers C.L.
      ,
      • Cantley L.C.
      ,
      • Datta S.R.
      • Brunet A.
      • Greenberg M.E.
      ). Cell growth and survival in a variety of tissues and cell types in response to IGF-1, insulin, and other growth factors is critically mediated by Akt (
      • Vivanco I.
      • Sawyers C.L.
      ,
      • Datta S.R.
      • Brunet A.
      • Greenberg M.E.
      ). Direct and indirect targets downstream of Akt include the mammalian target of rapamycin (mTOR), p70S6K, and PHAS-1 (4EBP-1), key regulatory proteins involved in translation and protein synthesis (
      • Rommel C.
      • Bodine S.C.
      • Clarke B.A.
      • Rossman R.
      • Nunez L.
      • Stitt T.N.
      • Yancopoulos G.D.
      • Glass D.J.
      ,
      • Cross D.A.
      • Alessi D.R.
      • Cohen P.
      • Andjelkovich M.
      • Hemmings B.A.
      ,
      • Nave B.T.
      • Ouwens M.
      • Withers D.J.
      • Alessi D.R.
      • Shepherd P.R.
      ,
      • Scott P.H.
      • Lawrence Jr., J.C.
      ).
      The PI3K/Akt pathway is a crucial intracellular signaling mechanism underlying muscle hypertrophy (
      • Glass D.J.
      ). In vivo activation of the PI3K pathway by introduction of a mutant form of Ras, competent only to activate PI3K, caused hypertrophy of regenerating muscle (
      • Murgia M.
      • Serrano A.L.
      • Calabria E.
      • Pallafacchina G.
      • Lomo T.
      • Schiaffino S.
      ), and pharmacological blockade of PI3K activity with the drug LY294002 (LY) blocks IGF-1-induced hypertrophy in vitro (
      • Rommel C.
      • Bodine S.C.
      • Clarke B.A.
      • Rossman R.
      • Nunez L.
      • Stitt T.N.
      • Yancopoulos G.D.
      • Glass D.J.
      ). During adaptive hypertrophy in adult muscle and in IGF-1-induced myotube hypertrophy, Akt is phosphorylated and activated, as is the Akt substrate mTOR (
      • Rommel C.
      • Bodine S.C.
      • Clarke B.A.
      • Rossman R.
      • Nunez L.
      • Stitt T.N.
      • Yancopoulos G.D.
      • Glass D.J.
      ,
      • Bodine S.C.
      • Stitt T.N.
      • Gonzalez M.
      • Kline W.O.
      • Stover G.L.
      • Bauerlein R.
      • Zlotchenko E.
      • Scrimgeour A.
      • Lawrence J.C.
      • Glass D.J.
      • Yancopoulos G.D.
      ,
      • Pallafacchina G.
      • Calabria E.
      • Serrano A.L.
      • Kalhovde J.M.
      • Schiaffino S.
      ). Additionally, hypertrophy elicits the phosphorylation of two known regulators of protein synthesis downstream of mTOR signaling, p70S6K and PHAS-1, thereby promoting increased translation (
      • Rommel C.
      • Bodine S.C.
      • Clarke B.A.
      • Rossman R.
      • Nunez L.
      • Stitt T.N.
      • Yancopoulos G.D.
      • Glass D.J.
      ,
      • Bodine S.C.
      • Stitt T.N.
      • Gonzalez M.
      • Kline W.O.
      • Stover G.L.
      • Bauerlein R.
      • Zlotchenko E.
      • Scrimgeour A.
      • Lawrence J.C.
      • Glass D.J.
      • Yancopoulos G.D.
      ,
      • Reynolds T.H.
      • Bodine S.C.
      • Lawrence Jr., J.C.
      ). The requirement for mTOR-mediated signaling in hypertrophy has been demonstrated pharmacologically; blockade with the mTOR inhibitor rapamycin decreases muscle hypertrophy in vivo and in vitro and blunts the hypertrophy-associated phosphorylation of p70S6K and PHAS-1 (
      • Rommel C.
      • Bodine S.C.
      • Clarke B.A.
      • Rossman R.
      • Nunez L.
      • Stitt T.N.
      • Yancopoulos G.D.
      • Glass D.J.
      ,
      • Bodine S.C.
      • Stitt T.N.
      • Gonzalez M.
      • Kline W.O.
      • Stover G.L.
      • Bauerlein R.
      • Zlotchenko E.
      • Scrimgeour A.
      • Lawrence J.C.
      • Glass D.J.
      • Yancopoulos G.D.
      ). Genetic approaches have provided further evidence for the role of the PI3K/Akt pathway in hypertrophy. Expression of constructs encoding constitutively active forms of either PI3K or Akt induced muscle hypertrophy both in vivo (
      • Bodine S.C.
      • Stitt T.N.
      • Gonzalez M.
      • Kline W.O.
      • Stover G.L.
      • Bauerlein R.
      • Zlotchenko E.
      • Scrimgeour A.
      • Lawrence J.C.
      • Glass D.J.
      • Yancopoulos G.D.
      ,
      • Pallafacchina G.
      • Calabria E.
      • Serrano A.L.
      • Kalhovde J.M.
      • Schiaffino S.
      ,
      • Lai K-M.
      • Gonzalez M.
      • Poueymirou W.T.
      • Kline W.O.
      • Na E.
      • Zlotchenko E.
      • Stitt T.N.
      • Economides A.
      • Yancopoulos G.D.
      • Glass D.J.
      ) and in vitro (
      • Rommel C.
      • Clarke B.A.
      • Zimmermann S.
      • Nunez L.
      • Rossman R.
      • Reid K.
      • Moelling K.
      • Yancopoulos G.D.
      • Glass D.J.
      ,
      • Rommel C.
      • Bodine S.C.
      • Clarke B.A.
      • Rossman R.
      • Nunez L.
      • Stitt T.N.
      • Yancopoulos G.D.
      • Glass D.J.
      ).
      Skeletal muscle atrophy, denoted by a decrease in muscle mass and fiber size, can be driven by such disparate stimuli as denervation, immobilization, sepsis, cachexia, or glucocorticoid treatment (
      • Jagoe R.T.
      • Goldberg A.L.
      ,
      • Jackman R.W.
      • Kandarian S.C.
      ). Atrophy is characterized by increases in protein degradation processes, particularly the ATP-dependent proteolytic ubiquitin-proteasome pathway (
      • Jagoe R.T.
      • Goldberg A.L.
      ). During atrophy, there is an increase in ubiquitin-protein conjugates and increased transcription of components of the ubiquitin degradation pathway (
      • Jagoe R.T.
      • Goldberg A.L.
      ,
      • Hasselgren P.O.
      ). A screen for genetic markers of atrophy identified two genes that are up-regulated rapidly in multiple models of muscle atrophy in vivo, including dexamethasone-induced wasting, which also show highly muscle-specific expression (
      • Gomes M.D.
      • Lecker S.H.
      • Jagoe R.T.
      • Navon A.
      • Goldberg A.L.
      ,
      • 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.
      • Pan Z.Q.
      • Valenzuela D.M.
      • DeChiara T.M.
      • Stitt T.N.
      • Yancopoulos G.D.
      • Glass D.J.
      ). These genes, MAFbx (muscle atrophy F-box, also called atrogin-1) and MuRF1 (muscle RING finger 1), both encode ubiquitin ligases, which function to conjugate ubiquitin to protein substrates (
      • Gomes M.D.
      • Lecker S.H.
      • Jagoe R.T.
      • Navon A.
      • Goldberg A.L.
      ,
      • 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.
      • Pan Z.Q.
      • Valenzuela D.M.
      • DeChiara T.M.
      • Stitt T.N.
      • Yancopoulos G.D.
      • Glass D.J.
      ). The functional importance of these gene products in atrophy processes was demonstrated by the generation of MAFbx–/– and MuRF1–/– mice. Mice lacking either gene show sparing of muscle mass following denervation (
      • 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.
      • Pan Z.Q.
      • Valenzuela D.M.
      • DeChiara T.M.
      • Stitt T.N.
      • Yancopoulos G.D.
      • Glass D.J.
      ). Furthermore, MuRF1 and MAFbx have been shown to be up-regulated in a number of other models of atrophy, indicating that these genes are highly faithful markers of the atrophy process (
      • Gomes M.D.
      • Lecker S.H.
      • Jagoe R.T.
      • Navon A.
      • Goldberg A.L.
      ,
      • 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.
      • Pan Z.Q.
      • Valenzuela D.M.
      • DeChiara T.M.
      • Stitt T.N.
      • Yancopoulos G.D.
      • Glass D.J.
      ,
      • Wray C.J.
      • Mammen J.M.
      • Hershko D.D.
      • Hasselgren P.O.
      ,
      • Li Y.-P.
      • Chen Y.
      • Li A.S.
      • Reid M.B.
      ).
      In recent studies it was shown that the hypertrophy-inducing PI3K/Akt pathway could dominantly block the atrophy-inducing effects of dexamethasone (
      • Sandri M.
      • Sandri C.
      • Gilbert A.
      • Skurk C.
      • Calabria E.
      • Picard A.
      • Walsh K.
      • Schiaffino S.
      • Lecker S.H.
      • Goldberg A.L.
      ,
      • Stitt T.N.
      • Drujan D.
      • Clarke B.A.
      • Panaro F.J.
      • Timofeyva Y.
      • Kline W.O.
      • Gonzalez M.
      • Yancopoulos G.D.
      • Glass D.J.
      ), via Akt-mediated phosphorylation and subsequent inhibition of the FOXO family of transcription factors (
      • Sandri M.
      • Sandri C.
      • Gilbert A.
      • Skurk C.
      • Calabria E.
      • Picard A.
      • Walsh K.
      • Schiaffino S.
      • Lecker S.H.
      • Goldberg A.L.
      ,
      • Stitt T.N.
      • Drujan D.
      • Clarke B.A.
      • Panaro F.J.
      • Timofeyva Y.
      • Kline W.O.
      • Gonzalez M.
      • Yancopoulos G.D.
      • Glass D.J.
      ); FOXO was shown to be necessary for the induction of both MuRF1 (
      • Stitt T.N.
      • Drujan D.
      • Clarke B.A.
      • Panaro F.J.
      • Timofeyva Y.
      • Kline W.O.
      • Gonzalez M.
      • Yancopoulos G.D.
      • Glass D.J.
      ) and MAFbx/atrogin-1 (
      • Sandri M.
      • Sandri C.
      • Gilbert A.
      • Skurk C.
      • Calabria E.
      • Picard A.
      • Walsh K.
      • Schiaffino S.
      • Lecker S.H.
      • Goldberg A.L.
      ,
      • Stitt T.N.
      • Drujan D.
      • Clarke B.A.
      • Panaro F.J.
      • Timofeyva Y.
      • Kline W.O.
      • Gonzalez M.
      • Yancopoulos G.D.
      • Glass D.J.
      ). Thereby by blocking FOXO, Akt blocked the induction of atrophy signaling.
      In an effort to further characterize hypertrophy/atrophy interactions, we sought to define genes that were induced in one direction by IGF-1 and inversely regulated by the atrophy-inducing glucocorticoid dexamethasone (DEX). These inversely regulated genes would comprise a set of markers of both hypertrophy and atrophy and would therefore function as barometers for the growth state of the muscle. We further sought to determine whether the IGF-1/PI3K/Akt pathway was dominant in regulating these markers (as it was in the case of MuRF1 and MAFbx) and, if so, which branches of the Akt pathway were involved in this instance of IGF-1-mediated regulation.

      EXPERIMENTAL PROCEDURES

      Cell Culture—C2C12 myoblasts (American Type Culture Collection (ATCC), Rockville, MD) were maintained in define media as described previously (
      • Rommel C.
      • Bodine S.C.
      • Clarke B.A.
      • Rossman R.
      • Nunez L.
      • Stitt T.N.
      • Yancopoulos G.D.
      • Glass D.J.
      ). The myoblasts were fused into myotubes at confluence, by shifting the proliferation medium (Dulbecco's modified Eagle's medium/10% fetal bovine serum) to differentiation media (Dulbecco's modified Eagle's medium/2% horse serum) and altering the atmospheric conditions from 5 to 7.5% CO2. The time point at which differentiation is induced is referred to as day 0 (D0). The concentration of chemicals used was 10 ng/ml IGF-1 (“long-R3-IGF-1,” Sigma), 100 μm DEX (Sigma), 10 μm LY294002 (Calbiochem), and 20 ng/ml rapamycin (RAP) (Calbiochem). IGF-1 and the pharmacological inhibitors LY and RAP were administered either on day two (D2) or day three (D3) post-fusion, while DEX treatments were administered on D3.
      Cultures were fixed and photographed by glutaraldehyde-induced autofluorescence, as described (
      • Stitt T.N.
      • Drujan D.
      • Clarke B.A.
      • Panaro F.J.
      • Timofeyva Y.
      • Kline W.O.
      • Gonzalez M.
      • Yancopoulos G.D.
      • Glass D.J.
      ). Myotube diameters were measured at the end of each treatment using SPOT RT v3.2 for Windows software. The results are expressed as means ± S.E.; statistical significance was assessed using a t test for paired samples.
      For FOXO1 immunolocalization, adenoviral infections were performed as described previously (
      • Stitt T.N.
      • Drujan D.
      • Clarke B.A.
      • Panaro F.J.
      • Timofeyva Y.
      • Kline W.O.
      • Gonzalez M.
      • Yancopoulos G.D.
      • Glass D.J.
      ). Briefly, myotube cultures in 6-well dishes were infected with myc-tagged wild-type (wt) or mutant, constitutively active FOXO1 adenovirus stocks, serum-starved for 20 h beginning on day 3 post-differentiation, and then treated with either 10 μm LY294002 or 20 ng/ml RAP for 3 h prior to fixation in 3.2% paraformaldehyde. Myotubes were permeabilized with 0.1% Nonidet P-40 and incubated with 9E10 anti-myc monoclonal antibody overnight, followed by 2 h of staining with Cy3-conjugated goat anti-mouse antibody (Sigma).
      Immunoblot Analysis—Cells were lysed in Triton buffer (0.5% Triton X-100, 250 mm NaCl, 50 mm Tris, pH 7.5, 1 mm EDTA, 50 mm NaF) with various protease inhibitors including 1 mm sodium orthovanadate, 1 μg/ml aprotinin, 100 nm okadaic acid, 5 μg/ml tosyl lysyl chloromethyl ketone, 10 μg/ml tosyl-l-phenylalanine chloromethyl ketone, 10 μg/ml soybean trypsin, and 1 mm phenylmethylsulfonyl fluoride and cleared by centrifugation at 16,000 × g for 15 min at 4 °C. Protein concentration was quantified using Pierce bicinchoninic acid (BCA®) assay kit.
      For immunoblot analysis, soluble fractions were separated in 10% Invitrogen precast tris-glycine gels using SDS-PAGE. Phosphorylation of Akt was determined with rabbit polyclonal anti-Akt and phospho-Akt (Ser473) antibodies (Cell Signaling). A rabbit polyclonal anti-peptide polyclonal antibody against MAFbx was generated (Open Biosystems), against amino acids 19–63 of rat MAFbx protein sequence. Other antibodies used in the study, including rabbit anti-p70S6K polyclonal antibody (Santa Cruz Biotechnology) and anti-phospho p70S6K (Thr421/Ser424) polyclonal antibody (Cell Signaling), were used.
      Northern Analysis—Northern blot analyses were performed as described previously (
      • Stitt T.N.
      • Drujan D.
      • Clarke B.A.
      • Panaro F.J.
      • Timofeyva Y.
      • Kline W.O.
      • Gonzalez M.
      • Yancopoulos G.D.
      • Glass D.J.
      ). The isolation of the total RNA was completed using Qiagen's Rneasy® midi kit.
      Microarray Analysis—The integrity of the total RNA was determined using Agilent Technologies 2100 Bioanalyzer and the RNA 6000 Lab-Chip® kit. Total RNA concentration was measured by light absorption characteristics using a Beckman DU®520 general purpose UV-visible spectrophotometer. Gene expression was first determined using Agilent's mouse cDNA microarray kit (catalog number G4104A). In a second microarray experiment, Agilent's mouse 22k oligonucleotide microarray kit (catalog number G4121A) was used. Chips were scanned using the Agilent DNA microarray scanner. Analysis of the scanned chips was carried out using Cluster/Treeview by Michael Eisen at Stanford University.
      TaqMan® Real-time Quantitative Reverse Transcriptase PCR—The cDNA sequences for MAFbx/atrogin-1, MuRF1, metallothionein-1 (MT-1), metallothionein-2 (MT-2), metallothionein-3 (MT-3), and proliferin (PLF), and parvalbumin (PV) are obtainable from GenBank™. PCR primers and TaqMan® fluorogenic probes were designed from the corresponding cDNA sequences using the Primer Express 1.5 software program (Applied Biosystems), and synthesis was performed by Applied Biosystems. PCR was performed with 25 ng of cDNA using TaqMan® RT reagents kit and TaqMan® PCR core reagents kit (Applied Biosystems). Each RNA sample had a control reaction without reverse transcriptase, to evaluate any genomic DNA contamination. A standard curve generated from known amounts of genomic DNA was used to determine the amount of each RNA. A glyceraldehyde-3-phosphate dehydrogenase control RNA served to determine that equal amounts of cDNA were used in the analysis. Units used in Figs. 3 and 6B are arbitrary, based on a standard curve for RNA level using genomic DNA; for example 20 = the level of signal obtained with the sample gene from 20 ng of genomic DNA.
      Figure thumbnail gr3
      Fig. 3Confirmation of inversely regulated genes after treatment with 10 ng/ml IGF-1 or 100 μm DEX in C2C12 myotubes. Gene regulation of MAFbx, MuRF1, metallothioneins (MT-1, MT-2, MT-3), PV, and PLF after indicated treatments assessed by TaqMan® real-time reverse transcriptase PCR. Each bar represents the standard deviation of triplicates of each sample. A, control myotubes were left untreated with IGF1 or DEX and were harvested at time points as indicated. For IGF-1 treatment, 10 ng/ml IGF-1 was used. Myotubes were allowed to differentiate for 2 days and then were treated for either 24 h (D2: 24h IGF1) or 48 h (D3: 48h IGF1); alternatively, myotubes were differentiated for 3 days and then treated with IGF1 for 24 h (D3: 24h IGF1). 100 μm DEX was used. Myotubes were allowed to differentiate for 3 days and then were treated for either 8 (D3: 8h DEX) or 24 h (D3: 24h DEX). B, DEX concentration curve from 1 to 100 μm. Units are arbitrary, based on a standard curve for RNA level using genomic DNA; for example, 20 = the level of signal obtained with the sample gene from 20 ng of genomic DNA.
      Figure thumbnail gr6
      Fig. 6A, requirement of the PI3K/Akt/mTOR pathway for IGF-1 mediated gene changes in PLF, PLF-2, PLF-3, PV, MT-2, and MAFbx. Red signifies up-regulation, and green signifies down-regulation. B, TaqMan® real-time reverse transcriptase PCR analysis of MT-1, MT-2, MAFbx, PV, and PLF after the indicated treatments. Each bar represents the standard deviation of triplicates of each sample. Units are arbitrary, based on a standard curve for RNA level using genomic DNA; for example 20 = the level of signal obtained with the sample gene from 20 ng of genomic DNA.

      RESULTS

      IGF-1 Causes Hypertrophy, and Dexamethasone Induces Atrophy, in C2C12 Myotubes—We have previously validated the in vitro C2C12 muscle cell line as a system to delineate the signaling pathways mediating IGF-1-induced myotube hypertrophy and demonstrated that such hypertrophy is induced by the activation of the PI3K/Akt pathway (
      • Rommel C.
      • Bodine S.C.
      • Clarke B.A.
      • Rossman R.
      • Nunez L.
      • Stitt T.N.
      • Yancopoulos G.D.
      • Glass D.J.
      ), a mechanism subsequently implicated in hypertrophy in vivo (
      • Bodine S.C.
      • Stitt T.N.
      • Gonzalez M.
      • Kline W.O.
      • Stover G.L.
      • Bauerlein R.
      • Zlotchenko E.
      • Scrimgeour A.
      • Lawrence J.C.
      • Glass D.J.
      • Yancopoulos G.D.
      ). Furthermore, we have established an analogous in vitro model of skeletal muscle atrophy, utilizing the cachectic glucocorticoid DEX (
      • Stitt T.N.
      • Drujan D.
      • Clarke B.A.
      • Panaro F.J.
      • Timofeyva Y.
      • Kline W.O.
      • Gonzalez M.
      • Yancopoulos G.D.
      • Glass D.J.
      ), which induces muscle wasting in vivo by inducing the ATP-dependent proteasome pathway (
      • Wing S.S.
      • Goldberg A.L.
      ,
      • Hong D.H.
      • Forsberg N.E.
      ) and which also causes decreased protein production in vitro (
      • Stitt T.N.
      • Drujan D.
      • Clarke B.A.
      • Panaro F.J.
      • Timofeyva Y.
      • Kline W.O.
      • Gonzalez M.
      • Yancopoulos G.D.
      • Glass D.J.
      ,
      • Hong D.H.
      • Forsberg N.E.
      ).
      Our initial goal was to determine the set of genes whose expression was perturbed at least 2-fold upon treatment with sufficient levels of IGF-1 to induce hypertrophy or sufficient concentrations of DEX so as to induce phenotypic atrophy. To induce hypertrophy, C2C12 myotubes were differentiated for 2 days (D2 myotubes) and treated with IGF-1 for 24 or 48 h. Analysis of myotubes after 48 h of IGF-1 treatment demonstrated an increase in myotube diameters of 226% (0.86 versus 0.38 relative units) (Fig. 1A). Treatment with IGF-1 also induced phosphorylation of Akt (Fig. 1B), as had been previously demonstrated in myotubes (
      • Rommel C.
      • Clarke B.A.
      • Zimmermann S.
      • Nunez L.
      • Rossman R.
      • Reid K.
      • Moelling K.
      • Yancopoulos G.D.
      • Glass D.J.
      ,
      • Rommel C.
      • Bodine S.C.
      • Clarke B.A.
      • Rossman R.
      • Nunez L.
      • Stitt T.N.
      • Yancopoulos G.D.
      • Glass D.J.
      ); such biochemical activation served as a positive control, establishing that the PI3K/Akt pathway had been activated in the myotubes that were later assessed for mRNA changes.
      Figure thumbnail gr1
      Fig. 1Effects of IGF-1 and DEX on differentiated C2C12 myotubes. C2C12 myoblasts were grown to confluence. At day 0, proliferation media was changed to differentiation media (DM). At D2 or D3 post-differentiation, media was changed once, with or without the indicated treatment. Samples in differentiation media alone served as controls for treated samples. A, representative fields of differentiated C2C12 myotubes. 10 ng/ml IGF-1 induces hypertrophy in C2C12 myotubes after 48 h of IGF-1 treatment (D2:48). 100 μm DEX induces atrophy in C2C12 myotubes after treatment for 24 h (D3:24). Mean myotube diameters are represented in a graph on the right and expressed in relative units (n = 15). B, 10 ng/ml IGF-1 induces phosphorylation of Akt in C2C12 myotubes; myotubes were differentiated for 2 days and then treated with IGF-1 for 24 h (D2:24) or 48 h (D2:48); alternatively, myotubes were differentiated for 3 days and then treated with IGF-1 for 24 h (D3:24). Immunoblot analysis was accomplished using an anti-phospho Akt (Ser473) antibody (pAkt); the ratio of pAkt to total Akt (Akt) was calculated after bands were quantitated by densitometry and represented by a graph on the right, in arbitrary units. An antibody that recognizes non-phosphorylated Akt was used to determine total Akt levels. C, DEX increases MAFbx protein levels in C2C12 myotubes. D3 myotubes were treated with DEX for either 8 (D3:8) or 24h(D3:24). For the immunoblot (IB), a polyclonal anti-peptide MAFbx antibody was used (see “Experimental Procedures”).
      To induce atrophy, D3 myotubes were treated with DEX for either 8 or 24 h. The myotubes were subsequently fixed and assayed for changes in myotube diameters. Addition of DEX resulted in a distinct atrophic phenotype (Fig. 1A), with a decrease in myotube diameter of 37% (0.24 versus 0.38 relative units) (Fig. 1B). As a biochemical control, protein levels of the E3 ubiquitin ligase MAFbx were assessed (Fig. 1C), a previously validated marker of skeletal muscle atrophy (
      • Gomes M.D.
      • Lecker S.H.
      • Jagoe R.T.
      • Navon A.
      • Goldberg A.L.
      ,
      • 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.
      • Pan Z.Q.
      • Valenzuela D.M.
      • DeChiara T.M.
      • Stitt T.N.
      • Yancopoulos G.D.
      • Glass D.J.
      ). MAFbx levels increased after both 8 and 24 h of DEX treatment (Fig. 1C). Duplicate cultures were used for assessment of DEX-induced mRNA changes.
      Microarray Analysis Identifies Over 500 Genes Significantly Regulated in IGF-induced Hypertrophy or DEX-induced Atrophy Models—Microarray analysis was performed to identify genes regulated during DEX-induced atrophy and IGF-1-induced hypertrophy. In this first microarray experiment, only genes whose expression changed at least 3-fold in two time points were accepted as being differentially regulated. In this first experiment a cDNA chip was used (see “Experimental Procedures”); 268 genes were found to be regulated by IGF-1 treatment, and 438 genes were regulated by DEX treatment (Fig. 2A).
      Figure thumbnail gr2
      Fig. 2Genes differentially expressed during in vitro hypertrophy and atrophy. A, total number of regulated genes and comparison of expressed sequence tags and known genes after treatment of C2C12 myotubes with 10 ng/ml IGF-1 or 100 μm DEX. B, inversely regulated genes after treatments with 10 ng/ml IGF-1 or 100 μm DEX after allowing myotubes to undergo differentiation for 2 days and subsequent treatment for 24 h (D2, 24h) or 48 h (D2, 48h) or after differentiation for 3 days and subsequent treatment for 8 h (D3, 8h) or for 24h(D3, 24h). Metallothioneins (MT-1, MT-2), MAFbx, proliferins, and parvalbumin were significantly and inversely regulated after IGF-1 versus DEX treatment. Red signifies up-regulation, green signifies down-regulation, and gray is used where there is missing data.
      Identification of Inversely Regulated Genes—We were interested in genes that were inversely regulated by the hypertrophic IGF-1 treatment as opposed to the atrophic dexamethasone treatment; these inversely regulated genes would seem to be more useful markers, since they could be used in either atrophy or hypertrophy models to help validate the phenotype on a molecular basis. Twenty-six of the regulated genes were shown to be inversely regulated in atrophy and hypertrophy, as illustrated using the Gene Expression Pattern Analysis Suite Version 1.0 (GEPAS) tool (Fig. 2B). Of these, we focused on the five genes with profiles that exhibited up-regulation or down-regulation of 4-fold or more for at least one time point in both atrophic and hypertrophic samples. One of the genes that was inversely regulated was MAFbx, a gene that had been previously shown to be up-regulated in atrophy (
      • Gomes M.D.
      • Lecker S.H.
      • Jagoe R.T.
      • Navon A.
      • Goldberg A.L.
      ,
      • 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.
      • Pan Z.Q.
      • Valenzuela D.M.
      • DeChiara T.M.
      • Stitt T.N.
      • Yancopoulos G.D.
      • Glass D.J.
      ). In addition to MAFbx, both metallothionein 1 (MT-1) and metallothionein 2 (MT-2) were down-regulated in IGF-1-induced myotube hypertrophy and up-regulated in DEX-induced myotube atrophy. MT-1 was found to be regulated more than 11-fold in both processes. MT-2 was found to be down-regulated more than 16-fold in hypertrophy,and up-regulated more than 19-fold in atrophy. In contrast, PLF was up-regulated more than 9-fold in hypertrophy and down-regulated 4-fold in atrophy. PV was also found to be inversely regulated; it was up-regulated 18-fold in hypertrophy and down-regulated 3-fold in atrophy.
      Analysis and Confirmation of Inversely Regulated Genes by TaqMan® and Immunoblot—To determine whether the microarray results were reproducible using a second mode of mRNA analysis, TaqMan®, a quantitative fluorescence based PCR technique (
      • Valenzuela D.
      • Murphy A.
      • Frendewey D.
      • Gale N.
      • Economides A.
      • Auerbach W.
      • Poueymirou W.
      • Adams N.
      • Rojas J.
      • Yasenchak J.
      • Chernomorsky R.
      • Boucher M.
      • Elsasser A.
      • Esau L.
      • Zheng J.
      • Griffiths J.
      • Wang X.
      • Su H.
      • Xue Y.
      • Dominguez M.
      • Noguera I.
      • Torres R.
      • Macdonald L.
      • Stewart A.
      • DeChiara T.
      • Yancopoulos G.
      ), was performed on the same mRNA samples as had been used in the microarray (Fig. 3). TaqMan® confirmed up-regulation of MAFbx after DEX treatment and down-regulation of MAFbx upon IGF-1 treatment, in comparison with untreated controls (Fig. 3, A and B). MuRF1, a second ligase that had been previously shown to be up-regulated in atrophy (
      • 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.
      • Pan Z.Q.
      • Valenzuela D.M.
      • DeChiara T.M.
      • Stitt T.N.
      • Yancopoulos G.D.
      • Glass D.J.
      ), was similarly up-regulated in this study but was not inversely down-regulated by IGF-1 to a significant degree (Fig. 3, A and B). TaqMan® showed that both MT-1 and MT-2 were significantly down-regulated in mRNA obtained from the IGF-1-treated myotubes, as compared with untreated controls. Furthermore, these genes were also shown to be up-regulated in response to DEX (Fig. 3, A and B). Unlike MT-1 and MT-2, MT-3 remained relatively constant throughout all time points for both models (data not shown). In contrast to MAFbx, MT-1, and MT-2, all of which were up-regulated in atrophy but down-regulated during hypertrophy, PLF and PV were found to be up-regulated upon treatment with IGF-1, in comparison with untreated controls, and down-regulated upon treatment with DEX (Fig. 3, A and B). A dose response was performed with DEX, demonstrating that most inversely regulated genes perturbed by 100 μm DEX are also regulated at lower doses (Fig. 3B).
      The PI3K/Akt/mTOR Pathway Mediates IGF-1-induced Myotube Hypertrophy—We have previously demonstrated that the IGF-1/PI3K/Akt/mTOR pathway mediates skeletal muscle hypertrophy in C2C12 myotubes by inducing protein synthesis pathways (
      • Rommel C.
      • Bodine S.C.
      • Clarke B.A.
      • Rossman R.
      • Nunez L.
      • Stitt T.N.
      • Yancopoulos G.D.
      • Glass D.J.
      ,
      • Bodine S.C.
      • Stitt T.N.
      • Gonzalez M.
      • Kline W.O.
      • Stover G.L.
      • Bauerlein R.
      • Zlotchenko E.
      • Scrimgeour A.
      • Lawrence J.C.
      • Glass D.J.
      • Yancopoulos G.D.
      ). We therefore sought to determine whether this pathway also played a role in IGF-1-induced gene transcription and specifically the inversely regulated genes we had identified as markers of skeletal muscle atrophy and hypertrophy. First, as a control, we assessed the phenotypic and biochemical effect of the PI3K inhibitor LY and the mTOR inhibitor RAP on the myotubes that would later be used for mRNA analysis. Myotubes that were differentiated for 2 days (D2) were treated with pharmacologic agents, and phenotypes were assessed 48 h later. In the absence of IGF-1 treatment, pharmacologic inhibition of PI3K using LY294002 or mTOR using RAP caused insignificant decreases in myotube size (Fig. 4A). IGF-1 treatment caused a greater than 2-fold increase in myotube diameter (0.39 versus 0.94), which was inhibited almost to base line by LY (Fig. 4A). RAP treatment blocked 80% of the hypertrophy induced by IGF-1 (Fig. 4A). The efficacy of the RAP and LY reagents used in the microarray experiments was demonstrated separately by immunoblot (Western) analysis; to do these signaling studies, IGF-1 treatment was for 15 min on prestarved myotubes as opposed to the longer treatments used to induce mRNA changes (Fig. 4B). LY blocked IGF-1-mediated activation of Akt; RAP, which acts downstream of Akt, did not block phosphorylation of Akt but did inhibit phosphorylation of p70S6K, which requires mTOR activation for its phosphorylation downstream of IGF-1 in skeletal myotubes (
      • Rommel C.
      • Bodine S.C.
      • Clarke B.A.
      • Rossman R.
      • Nunez L.
      • Stitt T.N.
      • Yancopoulos G.D.
      • Glass D.J.
      ) (Fig. 4B).
      Figure thumbnail gr4
      Fig. 4Effects of IGF-1 and pharmacological inhibitors on differentiated C2C12 myotubes. A, comparison of phenotypes of C2C12 myotubes after treatment with differentiation media alone (Control), 10 ng/ml IGF-1, 10 μm concentration of the PI3K inhibitor LY, 20 ng/ml RAP, and combinations of IGF-1 and pharmacologic inhibitors. Treatment began on D2 post-differentiation and lasted for 48 h. Representative fields of each sample are shown. Average myotube diameters are shown in relative units, and statistical significance is indicated versus IGF-1-treated myotubes (n = 20). B, effects of IGF-1 and pharmacological inhibitors on phosphorylation of Akt and p70S6K. To confirm that phosphorylation of Akt is only blocked by 10 μm LY, and phosphorylation of p70S6K is blocked by both 10 μm LY and 20 ng/ml RAP, a separate set of serum-starved myotubes were treated with the same media as above for 15 min. Immunoblot analysis with anti-phospho Akt Ser473 antibody and anti-phospho p70S6K Thr421/Ser424.
      Blockade of the PI3K/Akt/mTOR Pathway Inhibits Most Genes Activated by IGF-1—In a second microarray experiment, we monitored the expression profiles of 22,000 genes from C2C12 myotubes treated with IGF-1, LY, RAP, IGF-1 + LY, and IGF-1 + RAP. In this experiment, an oligonucleotide chip was used (see “Experimental Procedures”); the cut-off criteria using the oligonucleotide chip was genes that were regulated at least 2-fold. IGF-1 regulated 242 genes (Fig. 5); LY blocked 89.1% of the global changes induced by IGF-1, indicating that the PI3K pathway mediates most of the 242 transcriptional alterations induced by IGF-1 (Fig. 5). Of the IGF-1 regulated genes that were blocked by treatment with LY, all but 1% were also blocked when treated with IGF-1 plus RAP, suggesting that the Akt/mTOR pathway downstream of PI3K was additionally required to mediate transcription activation by IGF-1. As shown in Fig. 6A, all of the genes that were inversely regulated by IGF-1 as opposed to DEX required an intact PI3K/Akt/mTOR pathway, since blockade with either LY or RAP blocked the inverse regulation of IGF-1. This demonstrates that the PI3K/Akt/mTOR pathway is necessary for mediating mRNA changes of MAFbx, PLF, PLF-2, PLF-3, PV, and MT-2 during hypertrophy. In this particular microarray study we used an oligonucleotide array that lacked MT-1. The regulation of this gene, as well as the regulation of the genes identified by microarray, were confirmed by TaqMan® analyses (Fig. 6B).
      Figure thumbnail gr5
      Fig. 5Global expression profiles in differentiated C2C12 myotubes. Cultures were treated at day 2 post-differentiation with IGF-1 alone (lane 4) and in combination with 10 μm LY and 20 ng/ml RAP (lanes 5 and 6, respectively). mRNA was harvested 48 h later, at day 4 post-differentiation. Red signifies up-regulation, and green signifies down-regulation.
      We and others (
      • Sandri M.
      • Sandri C.
      • Gilbert A.
      • Skurk C.
      • Calabria E.
      • Picard A.
      • Walsh K.
      • Schiaffino S.
      • Lecker S.H.
      • Goldberg A.L.
      ,
      • Stitt T.N.
      • Drujan D.
      • Clarke B.A.
      • Panaro F.J.
      • Timofeyva Y.
      • Kline W.O.
      • Gonzalez M.
      • Yancopoulos G.D.
      • Glass D.J.
      ) have shown that the IGF-1/PI3K/Akt anti-atrophy activity requires the blockade of “Forkhead box” (FOXO) transcription factors. In myotubes, FOXO proteins reside in the cytoplasm when phosphorylated by Akt and translocate to the nucleus upon dephosphorylation. Using adenoviral constructs expressing myc-tagged versions of wild-type FOXO1 (wtFOXO1) and a triple mutant form of FOXO1 (caF-OXO1), which had its three Akt phosphorylation sites changed to alanine residues, the nonphosphorylatable caFOXO1 is exclusively nuclear (Fig. 7, top panel). Inhibition of PI3K activity by treatment with LY294002 induces strong nuclear translocation of wtFOXO1 (Fig. 7, middle panel). However, inactivation of mTOR by rapamycin had no effect in nuclear translocation (Fig. 7, bottom panel), indicating that the transcriptional effects of mTOR are not induced by perturbing FOXO1 and further implicating an as-yet-undescribed transcriptional mediator downstream of mTOR, which is additionally required for most IGF-1-mediated gene regulation.
      Figure thumbnail gr7
      Fig. 7Inhibition of PI3K, but not mTOR, allows FOXO1 translocation to the nucleus. C2C12 myotubes were infected with adenoviruses expressing myc-tagged FOXO1 (wtFOXO1; left panels) or constitutively active FOXO1 with mutations of the three Akt phosphorylation sites (caFOXO1; right panels). Myotubes were infected for 24 h starting at day 2 post-differentiation. Starting on day 3, myotubes were serum-starved for 20 h and then incubated with LY, RAP, or untreated (Control) for 3 h. FOXO1 localization was detected by immunostaining with anti-myc 9E10 antibody.

      DISCUSSION

      Skeletal muscle atrophy is accompanied by the induction of a distinct transcriptional program, characterized in particular by the up-regulation of two genes that encode E3 ubiquitin ligases, MuRF1 and MAFbx (
      • Glass D.J.
      ). In this study we have made use of an in vitro model of skeletal myotube atrophy to study genes that are inversely regulated by atrophy and hypertrophy. This in vitro atrophy model entails treatment of myotubes with the cachectic glucocorticoid, dexamethasone, which we demonstrate is sufficient to induce characteristics of the atrophy condition, a decrease in myotube size, and an induction of MuRF1 and MAFbx. In a previous study (
      • Stitt T.N.
      • Drujan D.
      • Clarke B.A.
      • Panaro F.J.
      • Timofeyva Y.
      • Kline W.O.
      • Gonzalez M.
      • Yancopoulos G.D.
      • Glass D.J.
      ), it was demonstrated that dexamethasone also induces a decrease in total protein content in the myotubes, again consistent with the increased protein turnover seen during atrophy.
      The converse of atrophy is hypertrophy, which is induced by an increase in protein synthesis and is characterized by an increase in muscle fiber size. In contrast to atrophy, however, high-fidelity markers of the hypertrophy condition have not been well validated. Previously, IGF-1-mediated myotube hypertrophy has been used as a model system to study the IGF-1/PI3K/Akt signal transduction pathways that mediate the increase in protein synthesis and thus muscle hypertrophy. By studying both atrophy and hypertrophy conditions simultaneously, the current study establishes a new set of regulated genes, those transcripts that are not only perturbed by an atrophy stimulus but that are inversely regulated during hypertrophy. This inversely regulated subset of genes would presumably constitute an even more reliable set of genetic markers than genes simply regulated by either atrophy or hypertrophy individually, since the ability to be regulated by the opposing conditions makes the mRNA profile of this group of genes a barometer of the growth state of the muscle.
      The inversely regulated genes characterized in this study include the previously identified atrophy marker MAFbx, which is up-regulated during atrophy; MAFbx is also down-regulated during hypertrophy. The inverse regulation of MAFbx by IGF-1 was also established in a previous study, which focused on MAFbx, MuRF1, and ubiquitin regulation by IGF-1 (
      • Sacheck J.M.
      • Ohtsuka A.
      • McLary S.C.
      • Goldberg A.L.
      ). In addition to MAFbx, a set of genes that were found to be inversely regulated were the metallothioneins, a family of proteins that carry Zinc (Zn2+). Metallothioneins were previously identified as atrophy markers (
      • Sacheck J.M.
      • Ohtsuka A.
      • McLary S.C.
      • Goldberg A.L.
      ,
      • Lecker S.H.
      • Jagoe R.T.
      • Gilbert A.
      • Gomes M.
      • Baracos V.
      • Bailey J.
      • Price S.R.
      • Mitch W.E.
      • Goldberg A.L.
      ). These findings suggest the possibility that Zn2+ might be part of a regulated signaling pathway that helps mediate the atrophy/hypertrophy balance in skeletal muscle. One obvious set of proteins that use Zn2+ and are involved in atrophy are the RING finger-containing ubiquitin ligases; however, we have no evidence currently that Zn2+ pools are limiting in any compartment of the cell, such that enhanced transport would help regulate the function of individual Zn2+-binding proteins.
      In a prior study, it was reported that inhibition of the PI3K signaling pathway was sufficient to induce MAFbx up-regulation (
      • Sacheck J.M.
      • Ohtsuka A.
      • McLary S.C.
      • Goldberg A.L.
      ). In a distinct study, myotubes were subjected to amino acid starvation conditions (
      • Sandri M.
      • Sandri C.
      • Gilbert A.
      • Skurk C.
      • Calabria E.
      • Picard A.
      • Walsh K.
      • Schiaffino S.
      • Lecker S.H.
      • Goldberg A.L.
      ). In our study, mRNA changes were assessed in the presence of differentiation media containing both amino acids and 2% horse serum as opposed to simply phosphate-buffered saline salt buffer. When myotubes have adequate nutritional support, an up-regulation of MuRF1 and MAFbx was not convincingly observed simply by pharmacologically inhibiting PI3K for 24 or 48 h; up-regulation of the atrophy markers MuRF1 and MAFbx at the time points studied required the treatment of dexamethasone. It is possible that simultaneous inhibition of the mTOR pathway and the PI3K pathway is sufficient to up-regulate MAFbx, since mTOR is regulated by the presence of amino acids (
      • Hara K.
      • Yonezawa K.
      • Weng Q.P.
      • Kozlowski M.T.
      • Belham C.
      • Avruch J.
      ).
      In addition to identifying the genes that are inversely regulated in atrophy and hypertrophy, this study identifies a signaling pathway downstream of IGF-1, which mediates the inverse regulation of atrophy-induced genes by IGF-1. IGF-1 induces the activation of the PI3K/Akt pathway, which, as mentioned above, causes an increase in protein translation via activation of p70S6K and inhibition of 4E-BP (also known as PHAS-1) (
      • Pullen N.
      • Dennis P.B.
      • Andjelkovic M.
      • Dufner A.
      • Kozma S.C.
      • Hemmings B.A.
      • Thomas G.
      ,
      • von Manteuffel S.R.
      • Gingras A.C.
      • Ming X.F.
      • Sonenberg N.
      • Thomas G.
      ,
      • Gingras A.C.
      • Kennedy S.G.
      • O'Leary M.A.
      • Sonenberg N.
      • Hay N.
      ,
      • Somwar R.
      • Sumitani S.
      • Taha C.
      • Sweeney G.
      • Klip A.
      ,
      • Gingras A.C.
      • Gygi S.P.
      • Raught B.
      • Polakiewicz R.D.
      • Abraham R.T.
      • Hoekstra M.F.
      • Aebersold R.
      • Sonenberg N.
      ). It was recently shown that in addition to its hypertrophic effects, Akt can dominantly inhibit induction of the atrophy genes MuRF1 and MAFbx by phosphorylating and thereby inhibiting the function of the FOXO family of transcription factors (
      • Sandri M.
      • Sandri C.
      • Gilbert A.
      • Skurk C.
      • Calabria E.
      • Picard A.
      • Walsh K.
      • Schiaffino S.
      • Lecker S.H.
      • Goldberg A.L.
      ,
      • Stitt T.N.
      • Drujan D.
      • Clarke B.A.
      • Panaro F.J.
      • Timofeyva Y.
      • Kline W.O.
      • Gonzalez M.
      • Yancopoulos G.D.
      • Glass D.J.
      ). This study is distinct from the previous work in that it focuses purely on IGF-1-induced changes, in the absence of a concurrent atrophy signal. In that context, the present study demonstrates an additional effect of the IGF-1/PI3K signaling pathway. Not only does inhibition at the level of PI3K block the ability of IGF to inversely regulate atrophy-induced genes, it also demonstrates that blockade at the level of mTOR, which is downstream of Akt, is sufficient to inhibit perturbation of most of the genes regulated by IGF-1 in myotubes. Given the broad set of distinct signal transduction pathways activated by IGF-1, it was quite surprising to find that inhibition of the PI3K/Akt/mTOR pathway was sufficient to block the vast majority of the genes normally regulated by IGF-1. This suggests a model in which transcription is integrated and cross-regulated by the distinct signaling pathways downstream of IGF-1 and in which blockade of mTOR is sufficient to block most IGF-induced changes in transcription. Teleologically, this model seems satisfying because it suggests that skeletal muscle has multiple check points that need to be appropriately set before protein synthesis can proceed; if Akt has been activated but the mTOR pathway is inhibited (perhaps via a lack of amino acids) then it would not be beneficial for the tissue to commence attempts at protein synthesis. Conversely, even if the nutritional milieu of the muscle is adequate to support hypertrophy (and thus mTOR signaling is patent), the muscle requires that FOXO also be excluded from the nucleus, and therefore growth factor signaling is integrated with nutritional inputs. The fact that FOXO translocation was not effected by treatment with rapamycin suggests the “check point” downstream of mTOR is a distinct, FOXO-independent, transcriptional program.
      Thus, in this study we demonstrate for the first time that the Akt/mTOR pathway is additionally required for much of the IGF-1 mediated transcriptional changes in skeletal muscle cells. It is important to point out that the situation may be different in the context of a simultaneous atrophy signal, such as dexamethasone. From a clinical perspective, the finding that IGF-1 can dominantly and inversely modulate key atrophy-induced genes via the PI3K/Akt pathway helps to further validate this pathway as a target for activation by anti-atrophy therapeutics.

      Acknowledgments

      We thank Drs. L. S. Schleifer, P. Roy Vagelos, and the Regeneron community for enthusiastic support. We thank Trevor Stitt and Ka-man-man Lai for useful conversations. We also thank Procter & Gamble Pharmaceuticals for their continued support. We are indebted to S. Staton, V. Lan, and B. Ephraim for expert graphics work.

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