Autophagic flux and oxidative capacity of skeletal muscles during acute starvation

©20

13 L

ande

s B

iosc

ienc

e. D

o no

t dis

tribu

te.

TranslaTional research PaPer

www.landesbioscience.com autophagy 1

autophagy 9:10, 1–17; october 2013; © 2013 landes Bioscience

TranslaTional research PaPer

*Correspondence to: Sabah NA Hussain; Email: [email protected]: 11/14/12; Revised: 07/26/13; Accepted: 07/29/13http://dx.doi.org/10.4161/auto.25955

autophagy is an important proteolytic pathway in skeletal muscles. The roles of muscle fiber type composition and oxidative capacity remain unknown in relation to autophagy. The diaphragm (Dia) is a fast-twitch muscle fiber with high oxidative capacity, the tibialis anterior (Ta) muscle is a fast-twitch muscle fiber with low oxidative capacity, and the soleus muscle (sol) is a slow-twitch muscle with high oxidative capacity. We hypothesized that oxidative capacity is a major determinant of autophagy in skeletal muscles. Following acute (24 h) starvation of adult c57/Bl6 mice, each muscle was assessed for autophagy and compared with controls. autophagy was measured by monitoring autophagic flux following leupeptin (20 mg/kg) or colchicine (0.4 mg/kg/day) injection. oxidative capacity was measured by monitoring citrate synthase activity. in control mice, autophagic flux values were significantly greater in the Ta than in the Dia and sol. in acutely starved mice, autophagic flux increased, most markedly in the Ta, and several key autophagy-related genes were significantly induced. in both control and starved mice, there was a negative linear correlation of autophagic flux with citrate synthase activity. starvation significantly induced aMPK phosphorylation and inhibited aKT and rPs6KB1 phosphorylation, again most markedly in the Ta. starvation induced Foxo1, Foxo3 and Foxo4 expression and attenuated the phosphorylation of their gene products. We conclude that both basal and starvation-induced autophagic flux are greater in skeletal muscles with low oxidative capacity as compared with those with high oxidative capacity and that this difference is mediated through selective activation of the aMPK pathway and inhibition of the aKT-MTor pathways.

Autophagic flux and oxidative capacity of skeletal muscles during acute starvation

Mahroo Mofarrahi,1† Yeting Guo,1† Jeffrey a haspel,2 augustine MK choi,2 elaine c Davis,3, Gilles Gouspillou,1 russell T hepple,1 richard Godin,4 Yan Burelle4 and sabah na hussain1

1Department of critical care Medicine; McGill University health centre and Meakins-christie laboratories; Department of Medicine; McGill University; Montréal, Québec, canada; 2Division of Pulmonary and critical care Medicine; Department of Medicine; Brigham and Women’s hospital; Boston, Ma Usa;

3Department of anatomy and cell Biology; McGill University; Montréal, Québec, canada; 4Faculty of Pharmacy; Université de Montréal; Montréal, Québec, canada

†These authors contributed equally to this work.

Keywords: autophagy, skeletal muscles, starvation, fiber-type composition, oxidative capacity

Abbreviations: ACTB, actin, beta; AKT, v-akt murine thymoma viral oncogene homolog; AMPK, 5́ AMP-activated protein kinase; ANOVA, analysis of variance; ATG4B, autophagy-related 4B, cysteine peptidase; ATG5, autophagy-related 5; ATG7,

autophagy-related 7; ATG12, autophagy-related 12; ATG13, autophagy-related 13; ATG14, autophagy-related 14; BCL2/PPP1R50, B-cell CLL/lymphoma 2; BCL2L11/BIM, BCL2-like 11 (apoptosis facilitator); BECN1, Beclin 1, autophagy related; BNIP3, BCL2/adenovirus E1B 19 kDa interacting protein 3; BNIP3L/NIX, BCL2/adenovirus E1B 19 kDa interacting protein

3-like; DIA, diaphragm; EDL, extensor digitorium longus; EDTA, ethylenediaminetetraacetic acid; DTT, dithioreitol; FBXO32/ATROGIN1, F-box protein 32; FOXO1, forkhead box O1; FOXO3, forkhead box O3; FOXO4, forkhead box O4; GABARAPL1,

GABA(A) receptor-associated protein like 1; GP, gastrocnemius-plantaris; i.p., intraperitoneal; LAMP2A, lysosomal-associated membrane protein 2A; MAP1LC3B/LC3B, microtubule-associated protein 1 light chain 3 beta; MTORC1, mechanistic target of

rapamycin complex 1; PARK2, parkinson protein 2, E3 ubiquitin protein ligase (parkin); PBS, phosphate-buffered saline; PIK3C3, phosphatidylinositol 3-kinase, catalytic subunit type 3; PMSF, phenylmethylsulfonyl fluoride; PVDF, polyvinylidene difluoride ; RB1CC1/FIP200, RB1-inducible coiled-coil 1; ROS, reactive oxygen species; RPS6, ribosomal protein S6; RPS6KB1, ribosomal protein S6 kinase, 70 kDa, polypeptide 1; SEM, standard error of the mean; SOD1, superoxide dismutase 1, soluble; SOL, soleus;

SQSTM1/p62, sequestosome 1; TA, tibialis anterior; TRAF6/RNF85, TNF receptor-associated factor 6, E3 ubiquitin protein ligase; TUBB, tubulin, beta class I; ULK1, unc-51 like autophagy activating kinase 1; UVRAG, UV radiation-resistance associated

©20

13 L

ande

s B

iosc

ienc

e. D

o no

t dis

tribu

te.

2 autophagy Volume 9 issue 10

Introduction

Skeletal muscle protein degradation is accomplished by four pathways: the calpain, caspase, ubiquitin-proteasome and autophagy-lysosome pathways. Calpains and caspases are involved in muscle protein degradation in catabolic conditions and operate by breaking down sarcomeric proteins.1,2 They degrade the bulk of muscle protein, which exists as actomyosin complexes, into the monomeric proteins actin and myosin. The ubiquitin-protease pathway is mostly responsible for myofilament protein degradation, particularly in response to steroid therapy, denervation and starvation.3 The autophagy-lysosome pathway has been the least studied. In this pathway, autophagy, a highly-conserved adaptive process, consists of lysosomal degradation of cytosolic components and organelles, regardless of the mechanism by which they are delivered to the lysosomes. In macroautophagy, proteins and organelles such as mitochondria are sequestered in double-membrane vesicles called autophagosomes and delivered to lysosomes for degradation and subsequent recycling.4

Although macroautophagy has been described in all cells, its role in skeletal muscle protein degradation has, for the most part, been ignored. Recently, however, a number of observations suggest that skeletal muscle autophagy is crucial to overall muscle health and function. These observations include the detection of autophagosomes within myofibrils and beneath the sarcolemma of normal skeletal muscles,5 significant inductions of autophagy in limb muscles in response to starvation and denervation,6 and the fact that autophagic contributions to total muscle protein degradation in response to starvation can be as high as those mediated by the proteasome.6,7 Furthermore, autophagy has been shown to be critically important to the maintenance of normal skeletal muscle homeostasis, as indicated by the progressive myopathy and abnormal metabolism that develops in transgenic mice in response to muscle-selective inhibition of autophagy.8,9 Although little is known of the mechanisms regulating autophagy in skeletal muscles, the observation that overexpression of a mutant form of superoxide dismutase 1 (SOD1) selectively triggers excessive autophagy suggests that oxidative stress is a major stimulus.10

Despite this recent progress in elucidating the functional roles of autophagy in the regulation of skeletal muscle mass and function, factors that determine the extent of autophagy in skeletal muscles remain unclear. One of these factors is fiber-type composition, and recent studies have focused upon delineating its role. An early report by Mizushima et al.5 suggests that a strong relationship between fiber type composition and autophagy exists in skeletal muscles of mice. These authors measured MAP1LC3B (hereafter, LC3B) protein staining (a marker of autophagosomes) in GFP-LC3 transgenic mice and found that acute starvation significantly induced levels of LC3B-labeled vesicles in fast-twitch extensor digitorum longus (EDL) muscles as compared with slow-twitch soleus (SOL) muscles.5 In a more recent study, Ogata et al.11 describes significant increases in the intensity of the lipidated form of LC3B protein in fast-twitch plantaris muscles of starved rats as compared with slow-twitch SOL. They concluded that the degree of starvation-induced autophagy in skeletal muscles

was related to fiber type composition. However, the conclusions of Mizushima et al. and Ogata et al. are based on comparisons between slow-twitch, high oxidative capacity muscle (SOL) and fast-twitch, low oxidative capacity muscles (EDL and plantaris). It is, therefore, difficult to ascertain from these studies whether or not fiber type composition alone exerts a discrete influence on the degree of autophagy in skeletal muscles, or if oxidative capacity per se might be a determining factor.

Oxidative capacity, a measure of the maximal capacity of a muscle to use oxygen, is linked to total volume of mitochondria and regulation of mitochondrial contents. The importance of determining the relationship between autophagy and oxidative capacity stems from recent studies demonstrating the existence of several mitochondria-derived signals, such as fusion-fission machinery, reactive oxygen species (ROS), and mitochondrial membrane potential, that trigger autophagic recycling of cytosolic proteins and mitochondria (mitophagy).12-14 The first objective of this study, therefore, is to investigate the relationship between degree of autophagy and oxidative capacity in skeletal muscles, both at the basal level and in response to acute starvation. To achieve this objective, we assessed autophagy in three different types of skeletal muscle: muscles that are fast-twitch with high oxidative capacity, muscles that are fast-twitch with low oxidative capacity; and muscles that are slow-twitch with high oxidative capacity. In the mouse, the DIA, tibialis anterior (TA) and soleus, respectively, represent such muscles. Indeed, mouse DIA has been shown to have relatively high proportions of type II fast-twitch fibers alongside relatively high mitochondrial content as compared with the TA and SOL (supplementary materials).15,16 In specific experiments, we also evaluated the gastrocnemius-plantaris (GP) and EDL as additional representatives of fast-twitch muscles with low oxidative capacity. A citrate synthase activity assay was used to measure oxidative capacity, as it has recently been shown to be an excellent biomarker of mitochondrial content in skeletal muscles.17

In the majority of studies that have evaluated the degree of autophagy in various skeletal muscles, investigators have relied on the concept of autophagic flux, or the entire process of autophagy from formation of a double-membraned vesicle to cargo degradation. Measuring the intensity of LC3B-II protein, a form of LC3B conjugated to phosphatidylethanolamine that is incorporated into the autophagosomes, is a quantitative index of autophagic flux. However, it has been well established that this method is inadequate to distinguish between impaired autophagosome degradation and enhanced autophagosome formation. Two recent reports have thus proposed new approaches to measuring in vivo autophagic flux in various organs. Haspel et al.18 and Ju et al.19 have used leupeptin and colchicine injection, respectively, to impair the degradation of autophagosomes by the lysosome and measured the resulting accumulation of autophagosome markers as an index of autophagic flux. In this study, we use both approaches to accurately quantify the degree of autophagy in muscles.

It is increasingly apparent that acute starvation induces a prolonged autophagy time course in skeletal muscles as compared with other organs.5,6 Several studies have suggested that sustained

©20

13 L

ande

s B

iosc

ienc

e. D

o no

t dis

tribu

te.


autophagy in starved skeletal muscles requires the activation of a nontranscriptional program initiated by the ULK1-ATG13-RB1CC1/FIP200 protein complex and a transcriptional program initiated, in part, by FOXO3 transcription factor. These programs are designed to upregulate the expression of several autophagy-related proteins that are then selectively degraded following autophagosome/lysosome fusion.4,6,7 One factor that has been identified as a regulator of ULK1-ATG13-RB1CC1 initiation of autophagy is the AMP-activated protein kinase (AMPK) pathway, which directly phosphorylates and activates ULK1. Another regulator is complex 1 of the mammalian target of rapamycin (MTORC1) activity, which phosphorylates and inhibits ULK1 and ATG13 proteins, thereby inhibiting autophagy.20-22 The extent to which activation of these regulatory programs occurs in various skeletal muscles in response to acute starvation are unclear. Although it has been shown that FOXO3 is involved, it remains unknown as to whether or not any of the other FOXO transcription factors are activated in starved skeletal muscles and whether their activation in response to acute starvation may be related to skeletal muscle oxidative capacity. Therefore, the second objective of this study is to investigate relationships between activation of nontranscriptional and transcriptional autophagy regulation and skeletal muscle oxidative capacity in response to acute starvation.

Results

Citrate synthase activityActivity of the mitochondrial enzyme citrate synthase (index

of oxidative capacity) in the DIA, TA, SOL and GP of control mice averaged 1320 ± 45, 818 ± 34, 1180 ± 48 and 566 ± 39 µmol/min/mg protein, respectively. These results show that the DIA exhibits the highest level of citrate synthase activity, followed by the SOL, GP and TA.

Basal autophagyFigure 1 represents mRNA levels of autophagy-related genes

in skeletal muscles of control mice. mRNA levels are expressed as fold change relative to that measured in the DIA (the muscle with the highest citrate synthase activity). Ulk1, Atg5 and Atg12 mRNA levels are significantly higher in the TA and GP muscles as compared with the DIA, while SOL levels are similar to those of the DIA. Expression of the BECN1 (BECLIN-1) complex (Becn1, Pik3c3, Uvrag and Atg14), Lc3b, Gabarapl1 and two important modulators of mitophagy, Bnip3 and Park2/Parkin, are significantly lower in the TA and GP as compared with the DIA, while SOL levels are similar or slightly higher than those of the DIA (Fig. 1). Expression of Atg7, Atg4b, the Becn1 complex inhibitor 1700021K19Rik/Rubicon, and the adaptor protein SQSTM1/p62 are similar in all muscles (Fig. 1). Figure 2 illustrates protein levels of various autophagy-related genes in skeletal muscles. ULK1, ATG5 and ATG12–ATG5 levels are significantly higher as compared with the DIA, while BECN1, PIK3C3, LC3B-II, BNIP3 (the dimer form), and PARK2 levels are significantly lower in the TA and GP as compared with the DIA. Levels of these proteins in the SOL are similar to those detected in the DIA.

Figure 3 shows representative examples of basal LC3B-II and SQSTM1 flux measurements using leupeptin and colchicine injections in control animals. Both substances promote the accumulation of LC3B-II and SQSTM1 proteins in skeletal muscles although the relative increase in LC3B-II protein intensity (autophagic flux) was significantly greater in the TA and GP muscles as compared with the DIA (Fig. 3A–C). That of the SOL is not statistically different from the DIA. Similarly, greater levels of SQSTM1 protein accumulation was observed in the TA and GP muscles in response to leupeptin and colchicine injections compared with the DIA and SOL muscles further confirming that autophagic flux in control mice is significantly higher in the TA and GP muscles compared with the DIA and SOL muscles (Fig. 3A–C).

Figure 1. mrna expression of various autophagy-related genes in the diaphragm (Dia), soleus (sol), tibialis anterior (Ta) and gastrocne-mius-plantaris (GP) muscles of control mice. Values (means ± seM) are expressed as fold change relative to Dia. *p < 0.05, as compared with Dia. TUBB: β-TUBUlin. n = 6 per group.

©20

13 L

ande

s B

iosc

ienc

e. D

o no

t dis

tribu

te.


Acute starvation-induced autophagyFigure 4 shows representative electron micrographs of

skeletal muscle samples from control and acutely starved mice. Also shown are mean values of the numbers of autophagosomes detected in these muscles in control and acutely starved (24 h) mice. In control mice, few autophagosomes can be visualized in the DIA muscle mainly in the subsarcolemmal region (Fig. 4). Relatively greater numbers of autophagosomes are detected in the TA and SOL compared with the DIA in control mice (Fig. 4). Acute starvation for 24 h elicits an increase in autophagosome formation in the DIA, TA and SOL muscles. Indeed, many autophagic vacuoles containing structurally abnormal sub-sarcolemmal and intermyofibrillar mitochondria can be clearly observed. The relative increase in autophagosome formation

in response to acute starvation is greater in the TA and SOL compared with the DIA (Fig. 4).

Acute starvation triggers significant increases in the mRNA expression of several autophagy-related genes, including Lc3b, Gabarapl1, Pik3c3, Atg7, Sqstm1, Bnip3, Park2 and Lamp2a (Fig. 5). Relative increases in autophagy-related gene expression are significantly greater in the TA and GP as compared with the DIA and SOL (Fig. 5). The autophagy-related genes Becn1, Atg12 and Atg4b are not induced by acute starvation (Fig. 5).

Immunoblotting of LC3B protein in all skeletal muscles confirmed that acute starvation for 24 h triggers significant increases in the intensity of lipidated LC3B protein (LC3B-II) and decreases in the intensity of free form of LC3B (LC3B-I) (Fig. 6A and B). Acute starvation for 24 h induces significantly larger

Figure 2. Protein expression of various autophagy-related genes. (A–C) representative immunoblots of various autophagy-related genes in Dia, Ta, sol, extensor digitorum longus (eDl) and GP muscles of control mice. (B–D) Protein optical densities of various autophagy-related genes in Dia, Ta, sol and GP muscles of control mice. Values (means ± seM) are expressed as fold change relative to Dia. *p < 0.05, as compared with Dia. acTB: β-acTin. n = 6 per group.

©20

13 L

ande

s B

iosc

ienc

e. D

o no

t dis

tribu

te.


accumulations of LC3B-II protein and significantly greater increase in the ratio of LC3B-II to LC3B-I in the TA, EDL and GP muscles as compared with the DIA (Fig. 6C and D). These differences in the intensity of LC3B protein lipidation are not related to the duration of starvation since 6 h and 48 h of

starvation also elicit significantly greater increase in LC3B lipidation in the TA muscle compared with the DIA and SOL (Fig. 6E and F). Acute starvation for 24 h triggers significant accumulations of LC3B-II protein in all skeletal muscles of starved animals when autophagic flux is measured using the

Figure 3. autophagic flux measurements in control mice. (A and B) representative immunoblots of lc3B and sQsTM1 proteins in Dia, Ta, sol and GP muscles of mice in the vehicle, leupeptin and colchicine injection subgroups. note relative increases in response to leupeptin and colchicine. (C) lc3B-ii and sQsTM1 autophagic fluxes in Dia, Ta, sol and GP muscles of control mice measured with the leupeptin and colchicine methods. *p < 0.05, as com-pared with Dia. n = 6 per group.

©20

13 L

ande

s B

iosc

ienc

e. D

o no

t dis

tribu

te.


leupeptin injection method, however, autophagic flux was significantly greater in the TA, GP and SOL compared with the DIA (Fig. 6G and H).

Figure 7 illustrates the relationships between LC3B-II autophagic flux in various skeletal muscles of control and starved animals. Both basal and acute starvation-induced autophagic flux is negatively related to the oxidative capacity of skeletal muscles.

Regulation of starvation-induced autophagy in skeletal muscles

Figure 8 demonstrates that phosphorylation of AMPKα on Thr172 is significantly induced in the DIA, TA and SOL although

the relative increase is significantly greater in the TA as compared with the DIA (Fig. 8). In response to acute starvation, AKT phosphorylation on Ser473 is significantly decreased in the TA muscle but remains unchanged in the DIA and SOL (Fig. 8). Phosphorylation of RPS6KB1 on Thr389 (MTORC1-specific activation site) significantly decreases in the TA but not in the DIA and SOL (Fig. 8). Similarly, phosphorylation of RPS6 (ribosomal protein S6) protein (target of MTORC1) and total RPS6 protein levels decline significantly in response to acute starvation (Fig. 8). These results indicate that acute starvation triggers a significant imbalance in the AMPK-AKT-MTORC1 axis, favoring enhanced AMPK activation and inhibition of the AKT and MTORC1 pathways. This imbalance is relatively greater in skeletal muscles with low oxidative capacity (such as the TA) than in those with high oxidative capacity (such as the DIA).

Figure 9 shows changes in total and phosphorylated ULK1 (Ser555) protein levels in the TA, DIA and SOL in control mice and mice exposed to 6, 24 or

48 h of starvation. In the TA, total ULK1 protein levels rise but phosphorylated ULK1 levels decline significantly compared with control values after 6 h of starvation (Fig. 9A and B). Both total and phosphorylated ULK1 protein levels rise significantly in response to 24 h and 48 h of starvation, however, the relative increase in phosphorylated ULK1 levels is less than that of total ULK1 and hence, the ratios of phosphorylated to total ULK1 protein levels decline significantly (Fig. 9C). In the DIA, both total ULK1 and phosphorylated ULK1 protein levels rise significantly in response to starvation, however, the relative increase in phosphorylated ULK1 levels is less than that of

Figure 4. Detection of autophagosomes using electron microscopy. (A, D and E) representative electron micrographs of auto-phagosomes (white arrows) in Dia, Ta and sol muscles, respectively, of acutely starved mice. Mito refers to the nucleus and mito-chondria, respectively. (B) representative electron micrographs of normal sub-sarco-lemmal and intermyofibrillar mitochondria in Dia of control mouse. (C) representative electron micrograph of an autophagosome in Ta of a control mouse. (F) autophagosome numbers in Dia, Ta and sol muscles of con-trol and acutely starved mice. Values (means ± seM) are expressed as the number of auto-phagosomes per field. n = 4 per group. *p < 0.05, as compared with Dia. #p < 0.05 com-pared with control.

©20

13 L

ande

s B

iosc

ienc

e. D

o no

t dis

tribu

te.


total ULK1 and hence, phosphorylated ULK1 to total ULK1 ratio decline significantly in response to starvation compared with control values (Fig. 9D–F). In the SOL, total ULK1 protein levels rise progressively in response to starvation while phosphorylated ULK1 levels rise significantly only after 6 h of starvation (Fig. 9G and H). Phosphorylated ULK1 to total ULK1 protein ratios decline progressively in the SOL in response to starvation (Fig. 9G–I).

Regulation of FOXO transcription factorsFigure 10 shows Foxo1, Foxo3 and Foxo4

mRNA levels in the DIA, TA and SOL of control and starved mice. In control animals, in all three muscles Foxo4 mRNA is more abundant than is either Foxo1 or Foxo3 mRNA (Fig. 10). Acute starvation elicits significant increases of Foxo1, Foxo3 and Foxo4 mRNA levels in each muscle although relative increases in the SOL are significantly smaller than in the DIA and TA (Fig. 10). Immunoblotting of proteins confirms inductions of both FOXO1 and FOXO3 (Fig. 11). Phosphorylated FOXO1 and FOXO3 protein levels are also elevated although ratios of phosphorylated to total FOXO1 or FOXO3 significantly declined to similar extents in all three muscles (Fig. 11). To assess the transcriptional activities of FOXO proteins, we detected mRNA levels of four genes which are known targets of FOXO transcription factors (Lc3b, Bnip3, Bnip3l/Nix and Fbxo32/Atrogin1).6,23,24 Acute starvation elicits significant increases in Lc3b (Fig. 5), Bnip3, Bnip3l and Fbxo32 mRNA levels in the DIA, TA and SOL muscles (Fig. 11) suggesting that FOXO transcriptional activities increase significantly in skeletal muscles in response to acute starvation.

Discussion

The main findings of this study are: )1) Basal autophagic flux measurements using leupeptin and colchicine injection methods yield quantitatively similar results and reveal that muscles with relatively high mitochondrial oxidative capacity have low basal autophagic flux while those with relatively low oxidative capacity have high basal autophagic flux; (2) Acute starvation elicits significant increases in skeletal muscle autophagic flux although relative increases are significantly greater in muscles with low oxidative capacity than in those with high oxidative capacity; (3) Acute starvation-induced autophagic flux in skeletal muscles is associated with activation of the AMPK pathway and inhibition of the AKT and MTORC1 pathways. This effect is more pronounced in muscles with low oxidative capacity; (4) Acute starvation triggers significant upregulation of mRNA levels of Foxo1, Foxo3 and Foxo4 transcription factors and inhibits their phosphorylation on residues that are targets of AKT. These changes in FOXO expression and phosphorylation are not related to skeletal muscle oxidative capacity.

Skeletal muscles significantly differ in their sensitivity to atrophic stimuli. Indeed, while starvation elicits significant atrophy and increased proteasome activity in fast-twitch muscles, unloading or inactivity elicits significantly greater degrees of atrophy in slow-twitch muscles.25 By measuring abundance of LC3B-II protein, previously published reports have concluded that starvation-induced autophagy in skeletal muscles is related to fiber-type composition and that fast-twitch muscles, such as the TA and EDL, develop significantly greater degrees of autophagy in response to starvation than do slow-twitch muscles, such as the SOL.5,11,26 This conclusion is problematic on two counts. First, it is based on using LC3B-II protein intensity as an index of autophagy although LC3B-II protein is both produced and degraded by autophagy. Therefore, measured levels do not reliably correlate with the proteolytic activity of the autophagy-lysosome system.27 Second, muscles differ not only in their fiber type composition but in their mitochondrial contents. This is clearly the case in mice where the DIA can be considered a fast-twitch muscle15,16 despite the fact that its mitochondrial contents are significantly greater than in the TA and GP, which are also fast-twitch muscles (supplementary materials).

To avoid the problem of using LC3B-II intensity alone as an index of autophagy, leupeptin and colchicine injections were used in this study to block autophagosome degradation. Measurements of LC3B-II protein accumulation were then performed on various skeletal muscles. This experimental approach has become the preferred method of autophagy evaluation in cell culture models and, more recently, in in-vivo conditions. Results reveal that leupeptin and colchicine injection both trigger significant accumulations of LC3B-II protein in various skeletal

Figure 5. mrna expression of various autophagy-related genes in Dia, Ta, sol and GP mus-cles of acutely starved mice. Gaba. refers to Gabarapl1. Values (means ± seM) are expressed as fold change relative to control group. #p < 0.05, as compared with control. n = 6 per group.

©20

13 L

ande

s B

iosc

ienc

e. D

o no

t dis

tribu

te.


muscles and that relative accumulations in a given muscle are similar when the two methods are compared. This suggests that either technique can be used to assess autophagic flux in murine skeletal muscles (Fig. 3).

This study reveals for the first time that autophagy-related genes are significantly differently expressed, depending on the muscle. We found that ULK1, ATG5 and ATG12 are more abundantly expressed in the TA, EDL and GP as compared

Figure 6. lc3B lipidation and autophagic flux measurements in response to starvation. (A) representative immunoblots of lc3B protein in Dia, Ta, sol, eDl and GP muscles of control and acutely (24 h) starved mice. Muscle samples were loaded on the same gel. (B) representative immunoblots of lc3B protein in Dia, Ta and sol muscles of control and acutely starved mice. samples were loaded on separate gels. (C and D) Protein optical densities of lc3B-ii and lc3B-ii/lc3B-i ratio in Dia, Ta, sol, eDl and GP muscles of acutely (24 h) starved mice. Values (means ± seM) are expressed as fold change relative to control group. *p < 0.05, as compared with Dia. #p < 0.05, as compared with control. n = 6 per group. (E) representative immunoblots of lc3B protein in Dia, Ta and sol muscles of control mice and mice exposed to 6 h and 48 h of starvation. (F) Protein optical densities of lc3B-ii protein in Dia, Ta and sol muscles obtained from mice exposed to 6 h and 48 h of starvation. Values (means ± seM) are expressed as fold change relative to control group. n = 4 per group. symbols are as described above. (G) representative immunoblots of lc3B in Dia, Ta and sol muscles of acutely starved mice by vehicle and leupeptin subgroup. note significant increase in lc3B-ii in response to leupeptin. (H) lc3B-ii autophagic flux values measured in Dia, Ta, sol and GP muscles of starved mice in response to leupeptin injection. *p < 0.05, as compared with Dia. n = 6 per group.

©20

13 L

ande

s B

iosc

ienc

e. D

o no

t dis

tribu

te.


with the DIA and SOL, whereas the BECN1 complex, LC3B, GABARAPL1, BNIP3 and PARK2 show a reverse pattern (Figs. 1 and 2). To our knowledge, this is the first report to document relative expressions of various autophagosomal components in a given organ. The mechanisms responsible for, and the functional implications of, this differential expression remain unclear. It is possible that the relatively high autophagic flux that is seen in muscles with low oxidative capacity, such as the TA, requires abundant ULK1 expression for rapid initiation of autophagosome formation. It is also likely that the relatively high presence of the BECN1 complex, LC3B, GABARAPL1, BNIP3 and PARK2 in muscles with high oxidative capacity, such as the DIA and SOL, reflects the fact that the majority of these proteins are required for recycling of the mitochondria by autophagosomes (mitophagy). In mitophagy, muscles with high mitochondrial contents and oxidative capacity require these proteins to maintain normal mitochondria quality control to a greater degree than do muscles with low mitochondria contents and oxidative capacity. It should be emphasized that abundance of autophagy-related proteins is not likely to be the only important factor in determining autophagic flux in skeletal muscles. Other important factors which influence the initiation of autophagy are post-translational modifications including phosphorylation of autophagy-related proteins. One such case is the phosphorylation of ULK1 and its regulation by various signaling pathways (see below). Another important factor that regulates initiation of autophagy is protein-protein interactions involving autophagy-related proteins. One such example is the interaction between BECN1 and other proteins such as BCL2 (also known as PPP1R50 in humans) and BCL2L11/BIM.28 Further studies are clearly needed to elucidate the importance of post-translational modifications and protein-protein interactions in determining the differences in autophagic flux among various skeletal muscles.

We also showed for the first time that basal and acute starvation-induced autophagy in skeletal muscles is negatively related to the oxidative capacity of muscle fibers. Mechanisms responsible for this relationship are not entirely clear. Autophagy

is regulated at several levels involving complex interactions between multiple signaling pathways. In this study, AMPK, AKT and MTORC1 pathway activations were assessed by measuring the phosphorylation of associated proteins or their downstream targets. In this study, AMPK activation was assessed by measuring Thr172 phosphorylation in the DIA, TA and SOL of control and starved mice. Phosphorylation of this residue by upstream kinases leads to AMPK activation.29 Results indicate that AMPK phosphorylation is significantly induced in skeletal muscles of starved mice, which is likely due to a food deprivation-induced increase in the cellular AMP/ATP ratio (Fig. 8). The degree of phosphorylation is substantially greater in the TA as compared with the DIA and SOL suggesting that AMPK is activated to a greater extent in the TA compared with the DIA and SOL. In this study, we assessed MTORC1 activation by measuring phosphorylation of RPS6KB1 and its substrate RPS6, both of which are effectors/downstream targets of the MTORC1 pathway that are required for protein synthesis and cell growth.30 We found that phosphorylation of RPS6KB1 and RPS6 is significantly attenuated in the TA, but not in the DIA or SOL of starved animals suggesting that starvation triggers significant inhibition of the MTORC1 mainly in TA but not in DIA and SOL muscles. The lack of effect of acute starvation on phosphorylation of AKT, RPS6KB1 and RPS6 in the DIA in our study differs from the observations of De Palma et al.31 and Grumati et al.32 who report significant AKT dephosphorylation and inactivation of the MTORC1 in the DIA of acutely starved mice. The reasons behind these differences are unclear. The AKT and MTORC1 pathways are influenced by numerous local and systemic factors such as aging, levels of growth factors, hormones, cytokines, chemokines, insulin, muscle activity, intracellular Ca2+, cAMP and reactive oxygen species.33 In addition, the response of AKT phosphorylation to starvation has recently been shown to be regulated by the E3 ligase TRAF6/RNF85.34 Finally, AKT activation in the DIA is particularly sensitive to stretching35 and differences in the ventilatory responses to starvation in various experiments may also account for the contradictory results in the sensitivity of skeletal muscle AKT and MTORC pathway to acute starvation.

Changes in AMPK and MTORC1 activities are likely to influence autophagy through specific actions on the autophagy initiation complex ULK1-ATG13-RB1CC1. There is evidence that phosphorylation status within the ULK1-ATG13-RB1CC1 complex dramatically changes with the nutrient status. Under normal nutrient supply conditions, MTORC1 associates with this complex through direct interactions between raptor and ULK1.36 In cultured cells, active MTORC1 phosphorylates ULK1 and ATG13 thereby inhibiting ULK1 kinase activity.36-38 Under starvation condition, MTORC1 activity is inhibited and both ULK1 and ATG13 are rapidly dephosphorylated. Shang et al.39 have recently identified 13 serine and threonine residues (including Ser556) in human ULK1 whose phosphorylation declines significantly in response to starvation or MTORC1 inhibition in cultured cells. Activation of ULK1 as a result of removal of the inhibitory effect of MTORC1 triggers ULK1-mediated activation of RB1CC1 and ATG13 followed by

Figure 7. relationship between lc3B-ii autophagic flux and citrate synthase activity in Dia, Ta, sol and GP muscles of control and acutely starved mice.

©20

13 L

ande

s B

iosc

ienc

e. D

o no

t dis

tribu

te.


Figure 8. see opposite page.

©20

13 L

ande

s B

iosc

ienc

e. D

o no

t dis

tribu

te.


Figure 8. activation of aMPK, aKT and MTorc1 pathways. (A) representative immunoblots of total and phosphorylated (Thr172) aMPK in Dia, Ta and sol muscles of control and acutely starved mice. (B) Protein optical densities of total and phosphorylated aMPK in Dia, Ta and sol muscles of control and acutely starved mice. Values (means ± seM) are expressed as fold change relative to control group. *p < 0.05, as compared with Dia. #p < 0.05, as compared with control. n = 6 per group. (C) representative immunoblots of total and phosphorylated (ser473) aKT in Dia, Ta and sol muscles of control and acutely starved mice. (D) Protein optical densities of total and phosphorylated (ser473) aKT in Dia, Ta and sol muscles of control and acutely starved mice. Values (means ± seM) are expressed as fold change relative to control group. #p < 0.05, as compared with control. n = 6 per group. (E) representative immunoblots of total and phosphorylated (Thr389) ribosomal protein s6 kinase B1 (rPs6KB1) in Dia, Ta and sol muscles of control and acutely starved mice. (F) Protein optical densities of total and phosphorylated (Thr389) rPs6KB1 in Dia, Ta and sol muscles of control and acutely starved mice. Values (means ± seM) are expressed as fold change relative to control groups. #p < 0.05, as compared with control. n = 6 per group. (G) representative immunoblots of total and phosphorylated (ser235/236) ribosomal protein s6 (rPs6) in Dia, Ta and sol muscles of control and acutely starved mice. (H) Protein optical densities of total and phosphorylated (ser235/236) rPs6 in Dia, Ta and sol muscles of control and acutely starved mice. Values (means ± seM) are expressed as fold change relative to control groups. #p < 0.05, as compared with control. n = 6 per group.

Figure 9. UlK1 expression and phosphorylation in response to starvation. (A, D and G) representative immunoblots of total and phosphorylated (ser555) UlK1 in Ta, Dia and sol muscles of control animals and those who underwent starvation for 6, 24 or 48 h. (B, E and H) Protein optical densities of total and phosphorylated UlK1 in Ta, Dia and sol muscles of control and acutely starved mice. Values (means ± seM) are expressed as fold change relative to control group. #p < 0.05, as compared with control. n = 4 per group. (C, F and I) ratios of optical densities of phosphorylated and total UlK1 proteins in Ta, Dia and sol muscles of control and acutely starved mice. Values are means ± seM #p < 0.05, as compared with control. n = 4 per group.

©20

13 L

ande

s B

iosc

ienc

e. D

o no

t dis

tribu

te.


translocation of the entire ULK1-RB1CC1-ATG13 complex to the phagophore membrane and activation of autophagy.37 Recent reports have confirmed that AMPK activates autophagy through two different mechanisms; direct phosphorylation of ULK1 by AMPK on residues including Ser317 and Ser777 residues.21 AMPK-mediated ULK1 phosphorylation on these residues triggers activation of ULK1 kinase activity.21 In addition, AMPK directly phosphorylates raptor and inhibits MTORC1 activation thereby removing the inhibitory effect of this complex on ULK1.40 In summary, ULK1 activation is controlled via a two-pronged mechanism that includes energy sensing (AMPK) and amino acid sensing (MTORC1). Our study reveals two important observations regarding the response of skeletal muscle ULK1 to acute starvation. First, ULK1 protein expression is significantly and progressively induced in the three muscles in response to acute starvation (Fig. 9). This observation suggests that sustained

autophagy in starved skeletal muscles requires enhanced transcription of ULK1 protein. Second, phosphorylation of ULK1 on Ser555 declines in response to acute starvation (Fig. 9). In murine ULK1, Ser555 residue is homologous to Ser556 in human ULK1 which is a target of MTORC1-mediated phosphorylation.39 Kim et al.21 have found that Ser555 of mouse ULK1 is not a target of AMPK-mediated phosphorylation. Relative dephosphorylation of ULK1 on Ser555 in starved skeletal muscles suggests that in vivo starvation triggers dissociation of the MTORC1 complex from ULK1 and activation of ULK1 kinase activity and autophagy initiation. The exact mechanisms involved in ULK1 dephosphorylation remain unclear. However, the fact that starvation-induced ULK1 dephosphorylation was observed in the TA, DIA and SOL despite the fact that MTORC1 inhibition was evident only in the TA suggests that AMPK is likely to be involved in ULK1 dephosphorylation. More importantly, our study reveals that upregulation of ULK1 expression and dephosphorylation of ULK1 doesn’t explain the differences in starvation-induced autophagic flux in skeletal muscles of low and high oxidative capacities because ULK1 upregulation and ULK1 dephosphorylation were observed in both types of muscles in response to acute starvation (Fig. 9).

Another likely mechanism underlying the strong relationship between autophagic flux and muscle oxidative capacity is mitochondrial production of reactive oxygen species (ROS). Several reports have confirmed that autophagy is activated by oxidative stress, which results from the production of O

2.-

and H2O

2 in response to nutrient starvation, growth factor

deprivation, or TNF exposure.41-43 ROS influence autophagy through several mechanisms of action including: (1) activation of AMPK and inhibition of MTORC1;44 (2) recruitment of BNIP3 to the mitochondria as a BCL2-binding competitor of BECN1, a trigger of autophagy;44 and (3) activation of the mitochondrial permeability transition pore, which results in loss of membrane potential and recruitment of PARK2 and autophagosomes to the mitochondria.45 Selective inhibition of mitochondrial ROS production strongly attenuates stress-induced autophagy, confirming the importance of mitochondria to the process.42,46 To evaluate whether or not the rate of mitochondrial ROS levels is related to muscle oxidative capacity, mitochondrial net H

2O

2 release from isolated DIA and TA of control mice was

measured. Results clearly indicate that mitochondrial H2O

2

levels are significantly greater in the TA as compared with the DIA (Fig. S1). This observation may be due to differences in rates of mitochondrial ROS production and/or elimination of ROS by endogenous oxidant systems. We propose that high mitochondrial ROS levels in skeletal muscles with low oxidative capacity predispose these muscles to increased autophagic flux in response to acute starvation.

It has been established in most tissues that acute starvation triggers transient autophagy that lasts for a few hours. In skeletal muscles, however, persistent generation of autophagosomes continues for days.5 A robust and a similar increase in LC3B-II protein intensity was observed in skeletal muscles in response to 6 h and 48 h of starvation (Fig. 6). It is unclear why the duration of starvation had no major influence on the relative

Figure 10. mrna expression of Foxo1, Foxo3 and Foxo4 transcription factors in Dia, Ta and sol muscles of control and acutely starved mice. Values (means ± seM) are expressed as copies per 104 copies of 18s. *p < 0.05, as compared with Dia. #p < 0.05, as compared with control. n = 6 per group.

©20

13 L

ande

s B

iosc

ienc

e. D

o no

t dis

tribu

te.


increase in skeletal muscle LC3B-II protein levels in our study. In rats, LC3B-II protein levels rose progressively in the plantaris muscle in response to 1, 2 and 3 d of starvation while significant increases in LC3B-II protein levels were observed in the SOL only after 3 d of starvation.11 In mice, Grumati et al.32 have noted a robust rise in skeletal muscle LC3B-II intensity after 24 h of starvation and that a mild increase in LC3B-II intensity is observed after 6 h and 12 h of starvation. The reasons behind the differences between these studies are unclear. We speculate that factors such as animal species, feeding protocol, age, initial weight and physical activities may influence the relationship between starvation duration and intensity of autophagy in skeletal muscles.

Acute starvation triggers significant inductions of several autophagy-related genes, such as those involved in autophagosome formation (LC3B, GABARAPL1, PIK3C3 and ATG7), mitophagy (BNIP3, PARK2 and SQSTM1), and chaperone-mediated autophagy (LAMP2A) (Fig. 5). The fact that these inductions are relatively more pronounced in muscles with high autophagic flux (TA and GP) than in those with low autophagic flux (DIA and SOL) concurs with the view that sustained autophagy in starved skeletal muscles requires the activation of transcriptional programs that are designed to maintain a steady supply of proteins that are integral to autophagosomes and that are recycled during autophagosome/lysosome fusion.4 The nature of these transcriptional programs has not as yet been clearly elucidated, except for the case of FOXO3, where several autophagy-related genes are known to be under its control. In starved adult murine skeletal muscles, for example, FOXO3 has been identified as an important component in the transcription of Lc3 and Bnip3 genes and overexpression of FOXO3 has been shown to be a sufficient activator of autophagy both in-vivo and in cultured muscle cells.6,7

Figure 10 indicates that acute starvation triggers significant inductions of Foxo1, Foxo3 and Foxo4 expression. Interestingly, these inductions are not related to oxidative capacity since the TA and DIA develop qualitatively similar inductions of these proteins. Inhibition of transcriptional activity occurs when proteins are targeted at specific residues for phosphorylation by AKT (FOXO1 at Ser256 and FOXO3 at Ser253). As phosphorylated FOXO proteins bind to 14–3-3 proteins in the cystosol, FOXO-dependent transcription is blocked. To evaluate whether or not activation of FOXO-mediated transcription might explain variations in autophagic flux in different skeletal muscles, we performed two types of measurements. First, we evaluated the phosphorylation status

of FOXO1 and FOXO3 proteins using immunoblotting. For FOXO1 and FOXO3, acute starvation significantly attenuates AKT-targeted phosphorylation of these factors and that this attenuation is not related to muscle oxidative capacity since it is quantitatively similar in the DIA, TA and SOL muscles (Fig. 11). Second, we evaluated transcriptional activities of FOXO proteins indirectly by measuring the expression of four of their known targets (Lc3b, Bnip3, Bnip3l and Fbxo32). Our results (Fig. 11) indicate that the expression of these targets of FOXO transcriptional activities rise significantly in the DIA, TA and SOL muscles in response to acute starvation, however, the relative increase in the expression of these targets is significantly greater in the TA than in the DIA and SOL muscles suggesting

Figure 11. expression and phosphorylation of FoXo1 and FoXo3 in response to starvation. (A) representative immunoblots of total and phosphorylated (ser256) FoXo1 in Dia, Ta and sol muscles of control and acutely starved mice. (B) Protein optical densities of total and phosphorylated (ser256) FoXo1 in Dia, Ta and sol muscles of control and acutely starved mice. Values (means ± seM) are expressed as fold change relative to control group. #p < 0.05, as compared with control. n = 6 per group. (C) representative immunoblots of total and phosphorylated (ser253) FoXo3 in Dia, Ta and sol muscles of control and acutely starved mice. (D) Protein optical densities of total and phosphorylated (ser253) FoXo3 in Dia, Ta and sol muscles of control and acutely starved mice. Values (means ± seM) are expressed as fold change relative to control group. #p < 0.05, as compared with control. n = 6 per group. (E) mrna expression of three targets of FoXo transcriptional activities (Bnip3, Bnip3l and Fbxo32) in Dia, Ta and sol of acutely starved mice. Values (means ± seM) are expressed as fold change relative to control group. #p < 0.05, as compared with control. n = 6 per group.

©20

13 L

ande

s B

iosc

ienc

e. D

o no

t dis

tribu

te.


that acute starvation induces significantly greater rise in FOXO transcriptional activities in the TA compared with the DIA and SOL muscles (Fig. 11). These results suggests that the enhanced autophagic flux that is seen in starved skeletal muscle is likely to be mediated in part by FOXO transcriptional activities and that increased FOXO expression and attenuated FOXO phosphorylation are both important factors in the regulation of autophagy in starved muscles. Given the complex array of genes that are involved in the autophagic process, we predict that there are probably other transcription factors that are also important to the differential regulation of autophagy-related genes in skeletal muscles with different oxidative capacities. Further research is required in order to confirm their roles.

In summary, we report here that autophagic flux correlates linearly and negatively with murine skeletal muscle oxidative capacity in control and acute starvation conditions. Induction of autophagy in low oxidative capacity muscle is associated with activation of the AMPK pathway and inhibition of the AKT and MTORC1 pathways. Acute starvation triggers significant inductions of Foxo1, Foxo3 and Foxo4, attenuates their phosphorylation and significantly enhances the expression of their target genes.

Materials and Methods

MaterialsAntibodies for LC3B (4108), BECN1 (3495), PIK3C3

(3358), ATG5 (8540), ATG12 (4180), ATG7 (8558), BNIP3L (12396), phospho-FOXO1 (Ser256)(9461), FOXO1 (2880), phospho-FOXO3 (Ser253)(9466), FOXO3 (2497), phospho-AKT (Thr308)(4056), AKT (4685), phospho-AMPKα (Thr172)(2535), AMPKα (2603), phospho-RPS6KB1 (Thr389)(9234), RPS6KB1 (2708), phospho-RPS6 (Ser235/236)(4858), RPS6 (2217), phospho-ULK1 (Ser555)(5869) and ULK1 (6439) were purchased from Cell Signaling Inc. (catalog numbers are shown in brackets). Antibody for SQSTM1/p62 was purchased from Abnova Inc. (M01). Antibodies for BNIP3 (B7931), TUBB/β-TUBULIN (T5201) and ACTB/β-ACTIN (A1978) were purchased from Sigma-Aldrich. Antibody for PARK2 (Prk8) was purchased from Santa Cruz Biotechnology Inc. (SC-32282). Pure LC3B protein was purchased from Enzo Life Sciences (ADI-APR-100)

Animal preparationAll procedures were approved by the Animal Care Committees

of McGill University and University of Montreal and were in accordance with the guidelines of the Canadian Council of Animal Care. Adult (8- to 12-wk-old) male wild-type C57/BL6j mice were fed ad libitum (control group) or subjected to 24 h of starvation (starved group). Animals were euthanized with sodium pentobarbital and the DIA, TA, SOL, GP and EDL were quickly excised, flash-frozen in liquid nitrogen, and stored at −80°C for later use.

Autophagic fluxTwo experimental approaches were used to assess basal and

acute starvation-induced autophagic flux in the DIA and limb muscles:

Leupeptin methodThis method involves the use of the protease inhibitor

leupeptin to monitor autophagic flux.Basal autophagic fluxMice were fed ad libitum and received a single intra-peritoneal

(i.p.) injection of phosphate buffered saline (PBS) (vehicle subgroup) or leupeptin (leupeptin subgroup, 20 mg/kg) 4 h prior to euthanasia. Skeletal muscles were excised and stored under -80° for later use.

Acute starvation-induced autophagic fluxMice were subjected to 24 h of starvation and received a single

i.p. injection of PBS (vehicle subgroup) or leupeptin (leupeptin subgroup, 20 mg/kg) 4 h prior to euthanasia. Skeletal muscles were excised and stored excised and stored under −80°C for later use.

Colchicine methodThis method involves the use of the microtubule depolarizing

agent colchicine to monitor autophagic flux.Basal autophagic fluxMice were fed ad libitum and received two i.p. injections

of PBS (vehicle subgroup) or colchicine (colchicine subgroup, 0.4 mg/kg/day). Twenty-four hours after the second PBS or colchicine injections, animals were euthanized and skeletal muscles were excised.

Acute starvation-induced autophagic fluxMice received two i.p. injections of PBS (vehicle subgroup) or

colchicine (colchicine subgroup, 0.4 mg/kg/day). Animals were then subjected to acute starvation for 24 h immediately following the second injection of PBS or colchicine and skeletal muscles were excised and stored for later use as described above.

Starvation time courseTo assess whether differences in the degree of autophagy

between various muscles are related to the duration of starvation, we also studied mice starved for 6 h and 48 h. Adult (8- to 12-wk-old) male wild-type C57/BL6j mice were fed ad libitum (control group) or subjected to 6 h or 48 h of starvation (starved group). Animals were euthanized with sodium pentobarbital and the DIA, TA and SOL were quickly excised, flash-frozen in liquid nitrogen and stored at −80°C for later use.

Electron microscopyMice were fed ad libitum (control group) or subjected to 24

h of starvation (starvation group). Animals were anesthetized with sodium pentobarbital and perfused at a pressure in the physiological range through the left ventricle with normal saline followed by 3% glutaraldehyde buffered with 0.1 M sodium cacodylate (pH 7.4). Following perfusion, DIA, TA and SOL muscles were excised, dissected into segments and placed in fresh fixative at room temperature for an additional 2 h. Following fixation, tissues were washed overnight in cacodylate buffer at 4°C then sequentially treated with 1% OsO

4 in buffer, 2%

tannic acid in buffer and 2% uranyl acetate in distilled water. Tissues were then dehydrated with a graded series of solutions from methanol to propylene oxide, infiltrated and embedded in EPON™, as previously described.47 Thin sections (60 nm) were counterstained with methanolic uranyl acetate and lead citrate and viewed using a FEI Tecnai™ 12 transmission

©20

13 L

ande

s B

iosc

ienc

e. D

o no

t dis

tribu

te.


electron microscope at 120 kV. Images were digitally captured. Morphometric quantification of autophagosomes (defined as vesicular compartments with double-limiting membranes containing heterogenous cytosolic materials or mitochondria) in the DIA and TA muscles was achieved by visualizing 60 random fields per muscle sample.

RNA extraction and real-time PCRTotal RNA was extracted from DIA and limb muscles using

a GenElute™ Mammalian Total RNA Miniprep Kit (Sigma-Aldrich, MRN10). Quantification and purity of total RNA was assessed by A260/A280 absorption. Total RNA (2 µg) was then reverse transcribed using a Superscript II® Reverse Transcriptase Kit and random primers (Invitrogen Canada, Inc. 18080-093). Reactions were incubated at 42°C for 50 min and at 90°C for 5min. Real-time PCR detection of mRNA expression was performed using a Prism® 7000 Sequence Detection System (Applied Biosystems). mRNA expression of autophagy-related genes were assessed due to their importance to (1) the initial phase of autophagosome formation (Ulk1, Becn1, Pik3c3/Vps34, Uvrag, Atg14 and 1700021K19Rik/Rubicon, (2) the expansion of the phagophore (Lc3b, Gabrarapl1 and Atg4b), (3) vesicle elongation (Lc3b and Atg12), (4) chaperone-mediated autophagy (Lamp2a), (5) genes involved in mitophagy (Sqstm1 and Park2) and (6) targets of FOXO transcription factors (Lc3b, Bnip3, Bnip3l, Fbxo32). Specific sets of primers that were used to detect these genes are listed in the supplementary materials. In all assays, 1.0 µl of reverse-transcriptase reagent was added to 25 µl of SYBR® Green master mix (Qiagen Inc., 330520) and 3.5 µl of 10 µM primer. The thermal profile used was as follows: 95°C for 10 min; 40 cycles each of 95°C for 15 s; 57°C for 30 s; and 72°C for 33 s. All real-time PCR experiments were performed in triplicate. A melt analysis for each PCR experiment was performed to assess primer-dimer formation or contamination. Relative mRNA level quantifications of target genes were determined using the threshold cycle (ΔΔC

T) method using the housekeeping gene

Actb. For the detection of Foxo1 (Mm00490671_m1), Foxo3 (Mm01185722_m1) and Foxo4 (Mm00840140_g1) mRNA levels, TaqMan® Gene Expression Assays (Applied Biosystems Inc.) were used. As a control, 18S levels were detected. TaqMan® master mix (4370048) was used in these assays and absolute copies of Foxo1, Foxo3, Foxo4 and 18S transcripts were derived from standard curves relating cycle threshold values of a given gene to copy numbers, as described.48 Gene expression values are given as copies per 103 copies of 18S.

Citrate synthase activity assayCitrate synthase activity was measured in the DIA and limb

muscles using a previously described method.49 All samples were homogenized in a 200 mM Tris extraction buffer containing 50 mM triethanolamine and 1mM EDTA at pH 7.4. Activity was measured by detecting the increase in absorbance at 412 nm of 200 µl reaction buffer (200 mM Tris, pH 7.4) containing 2 µm acetyl-CoA, 200 µm 5,5́ -dithiobis-(2-nitrobenzoic acid) (DTNB), 350 µm oxaloacetic acid and 0.1% Triton-x in a 96-well plate at 30°C. A molar extinction coefficient of 13.6 L mol−1 cm−1 for DTNB was used.

Protein extraction and immunoblottingFrozen muscle samples were homogenized in homogenization

buffer (10 mM tris-maleate, 3 mM EGTA, 275 mM sucrose, 0.1 mM DTT, 2 µg/ml leupeptin, 100 µg/ml PMSF, 2 µg/ml aprotinin and 1 mg/100 ml pepstatin A, pH 7.2). Samples were centrifuged at 1,000 g for 10 min. Pellets were discarded and supernatants were designated as crude homogenate. Total muscle protein levels in each sample were determined using the Bradford protein assay technique. Crude homogenate samples (25 µg/sample) were mixed with SDS sample buffer, boiled for 5 min at 95°C, then loaded onto tris-glycine sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE) and separated by electrophoresis. Proteins were transferred by electrophoresis to polyvinylidene difluoride (PVDF) membranes and blocked with 1% bovine serum albumin or milk for 1 h at room temperature. PVDF membranes were incubated overnight with primary antibodies at 4°C then washed and incubated with horseradish peroxidase-conjugated secondary antibody. Specific proteins were detected with an enhanced chemiluminescence kit (Millipore, WBLUC0100). Equal loading of proteins was confirmed by stripping membranes and reprobing with anti-TUBB or ACTB antibodies. Blots were scanned with an imaging densitometer and optical densities (OD) of protein bands were quantified using ImagePro® Plus software (Media Cybernetics). To quantify LC3B levels, a standard curve consisting of purified LC3B protein run alongside in the same gels (range 0.4 to 1.2 ng protein per lane). LC3B protein density was converted to LC3B protein quantity by extrapolation from the standard curve using regression analysis tools. These values were then normalized to mg of total muscle protein loaded per lane. LC3B autophagic flux was quantified as the difference in LC3B-II protein quantify on immunoblots obtained in the presence vs. the absence of leupeptin or colchicine. Similarly, differences in the optical densities of SQSTM1 in the presence and absence of leupeptin were quantified as index of SQSTM1 autophagic flux.

Statistical analysisResults are expressed as means ± SEM. Statistical differences

in autophagic flux, mRNA and protein levels within and between different skeletal muscles were analyzed using one-way ANOVA (Statistica Software, StatSoft, Inc.). Tukey post-hoc tests were used to identify significant differences (p < 0.05).

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

This work was funded by a grant awarded to SNAH from the Canadian Institutes of Health Research (CIHR) and by a grant awarded to SNAH from the Natural Sciences and Engineering Research Council of Canada (NSERC).

Supplemental Materials

Supplemental materials may be found here: www.landesbioscience.com/journals/autophagy/article/25955

©20

13 L

ande

s B

iosc

ienc

e. D

o no

t dis

tribu

te.


References1. Bartoli M, Richard I. Calpains in muscle wasting.

Int J Biochem Cell Biol 2005; 37:2115-33; PMID:16125114; http://dx.doi.org/10.1016/j.biocel.2004.12.012

2. Supinski GS, Callahan LA. Caspase activation contributes to endotoxin-induced diaphragm weakness. J Appl Physiol 2006; 100:1770-7; PMID:16484358; http://dx.doi.org/10.1152/japplphysiol.01288.2005

3. Diaz PT, Julian MW, Wewers MD, Clanton TL. Tumor necrosis factor and endotoxin do not directly affect in vitro diaphragm function. Am Rev Respir Dis 1993; 148:281-7; PMID:8342889; http://dx.doi.org/10.1164/ajrccm/148.2.281

4. Sandri M. Autophagy in skeletal muscle. FEBS Lett 2010; 584:1411-6; PMID:20132819; http://dx.doi.org/10.1016/j.febslet.2010.01.056

5. Mizushima N, Yamamoto A, Matsui M, Yoshimori T, Ohsumi Y. In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker. Mol Biol Cell 2004; 15:1101-11; PMID:14699058; http://dx.doi.org/10.1091/mbc.E03-09-0704

6. Mammucari C, Milan G, Romanello V, Masiero E, Rudolf R, Del Piccolo P, Burden SJ, Di Lisi R, Sandri C, Zhao J, et al. FoxO3 controls autophagy in skeletal muscle in vivo. Cell Metab 2007; 6:458-71; PMID:18054315; http://dx.doi.org/10.1016/j.cmet.2007.11.001

7. Zhao J, Brault JJ, Schild A, Cao P, Sandri M, Schiaffino S, Lecker SH, Goldberg AL. FoxO3 coordinately activates protein degradation by the autophagic/lysosomal and proteasomal pathways in atrophying muscle cells. Cell Metab 2007; 6:472-83; PMID:18054316; http://dx.doi.org/10.1016/j.cmet.2007.11.004

8. Moresi V, Carrer M, Grueter CE, Rifki OF, Shelton JM, Richardson JA, Bassel-Duby R, Olson EN. Histone deacetylases 1 and 2 regulate autophagy flux and skeletal muscle homeostasis in mice. Proc Natl Acad Sci U S A 2012; 109:1649-54; PMID:22307625; http://dx.doi.org/10.1073/pnas.1121159109

9. Masiero E, Agatea L, Mammucari C, Blaauw B, Loro E, Komatsu M, Metzger D, Reggiani C, Schiaffino S, Sandri M. Autophagy is required to maintain muscle mass. Cell Metab 2009; 10:507-15; PMID:19945408; http://dx.doi.org/10.1016/j.cmet.2009.10.008

10. Dobrowolny G, Aucello M, Rizzuto E, Beccafico S, Mammucari C, Boncompagni S, Belia S, Wannenes F, Nicoletti C, Del Prete Z, et al. Skeletal muscle is a primary target of SOD1G93A-mediated toxicity. Cell Metab 2008; 8:425-36; PMID:19046573; http://dx.doi.org/10.1016/j.cmet.2008.09.002

11. Ogata T, Oishi Y, Higuchi M, Muraoka I. Fasting-related autophagic response in slow- and fast-twitch skeletal muscle. Biochem Biophys Res Commun 2010; 394:136-40; PMID:20184860; http://dx.doi.org/10.1016/j.bbrc.2010.02.130

12. Narendra DP, Youle RJ. Targeting mitochondrial dysfunction: role for PINK1 and Parkin in mitochondrial quality control. Antioxid Redox Signal 2011; 14:1929-38; PMID:21194381; http://dx.doi.org/10.1089/ars.2010.3799

13. Scherz-Shouval R, Elazar Z. ROS, mitochondria and the regulation of autophagy. Trends Cell Biol 2007; 17:422-7; PMID:17804237; http://dx.doi.org/10.1016/j.tcb.2007.07.009

14. Romanello V, Guadagnin E, Gomes L, Roder I, Sandri C, Petersen Y, Milan G, Masiero E, Del Piccolo P, Foretz M, et al. Mitochondrial fission and remodelling contributes to muscle atrophy. EMBO J 2010; 29:1774-85; PMID:20400940; http://dx.doi.org/10.1038/emboj.2010.60

15. Augusto V, Padovani CR, Campos GER. Skeletal muscle fiber types in C57BL6J mice. Braz J Morphol Sci 2004; 21:89-94

16. Brown JD, Hancock CR, Mongillo AD, Benjamin Barton J, DiGiovanni RA, Parcell AC, Winder WW, Thomson DM. Effect of LKB1 deficiency on mitochondrial content, fibre type and muscle performance in the mouse diaphragm. Acta Physiol (Oxf) 2011; 201:457-66; PMID:21073663; http://dx.doi.org/10.1111/j.1748-1716.2010.02226.x

17. Larsen S, Nielsen J, Hansen CN, Nielsen LB, Wibrand F, Stride N, Schroder HD, Boushel R, Helge JW, Dela F, et al. Biomarkers of mitochondrial content in skeletal muscle of healthy young human subjects. J Physiol 2012; 590:3349-60; PMID:22586215; http://dx.doi.org/10.1113/jphysiol.2012.230185

18. Haspel J, Shaik RS, Ifedigbo E, Nakahira K, Dolinay T, Englert JA, Choi AM. Characterization of macroautophagic flux in vivo using a leupeptin-based assay. Autophagy 2011; 7:629-42; PMID:21460622; http://dx.doi.org/10.4161/auto.7.6.15100

19. Ju JS, Varadhachary AS, Miller SE, Weihl CC. Quantitation of “autophagic flux” in mature skeletal muscle. Autophagy 2010; 6:929-35; PMID:20657169; http://dx.doi.org/10.4161/auto.6.7.12785

20. Egan DF, Shackelford DB, Mihaylova MM, Gelino S, Kohnz RA, Mair W, Vasquez DS, Joshi A, Gwinn DM, Taylor R, et al. Phosphorylation of ULK1 (hATG1) by AMP-activated protein kinase connects energy sensing to mitophagy. Science 2011; 331:456-61; PMID:21205641; http://dx.doi.org/10.1126/science.1196371

21. Kim J, Kundu M, Viollet B, Guan K-L. AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Nat Cell Biol 2011; 13:132-41; PMID:21258367; http://dx.doi.org/10.1038/ncb2152

22. Alers S, Löffler AS, Wesselborg S, Stork B. Role of AMPK-mTOR-Ulk1/2 in the regulation of autophagy: cross talk, shortcuts, and feedbacks. Mol Cell Biol 2012; 32:2-11; PMID:22025673; http://dx.doi.org/10.1128/MCB.06159-11

23. Real PJ, Benito A, Cuevas J, Berciano MT, de Juan A, Coffer P, Gomez-Roman J, Lafarga M, Lopez-Vega JM, Fernandez-Luna JL. Blockade of epidermal growth factor receptors chemosensitizes breast cancer cells through up-regulation of Bnip3L. Cancer Res 2005; 65:8151-7; PMID:16166289; http://dx.doi.org/10.1158/0008-5472.CAN-05-1134

24. Lam EW, Francis RE, Petkovic M. FOXO transcription factors: key regulators of cell fate. Biochem Soc Trans 2006; 34:722-6; PMID:17052182; http://dx.doi.org/10.1042/BST0340722

25. Allen DL, Cleary AS, Lindsay SF, Loh AS, Reed JM. Myostatin expression is increased by food deprivation in a muscle-specific manner and contributes to muscle atrophy during prolonged food deprivation in mice. J Appl Physiol 2010; 109:692-701; PMID:20595541; http://dx.doi.org/10.1152/japplphysiol.00504.2010

26. Yamada E, Bastie CC, Koga H, Wang Y, Cuervo AM, Pessin JE. Mouse skeletal muscle fiber-type-specific macroautophagy and muscle wasting are regulated by a Fyn/STAT3/Vps34 signaling pathway. Cell Rep 2012; 1:557-69; PMID:22745922; http://dx.doi.org/10.1016/j.celrep.2012.03.014

27. Tanida I, Minematsu-Ikeguchi N, Ueno T, Kominami E. Lysosomal turnover, but not a cellular level, of endogenous LC3 is a marker for autophagy. Autophagy 2005; 1:84-91; PMID:16874052; http://dx.doi.org/10.4161/auto.1.2.1697

28. Luo S, Garcia-Arencibia M, Zhao R, Puri C, Toh PP, Sadiq O, Rubinsztein DC. Bim inhibits autophagy by recruiting Beclin 1 to microtubules. Mol Cell 2012; 47:359-70; PMID:22742832; http://dx.doi.org/10.1016/j.molcel.2012.05.040

29. Hawley SA, Davison M, Woods A, Davies SP, Beri RK, Carling D, Hardie DG. Characterization of the AMP-activated protein kinase kinase from rat liver and identification of threonine 172 as the major site at which it phosphorylates AMP-activated protein kinase. J Biol Chem 1996; 271:27879-87; PMID:8910387; http://dx.doi.org/10.1074/jbc.271.44.27879

30. Pullen N, Thomas G. The modular phosphorylation and activation of p70s6k. FEBS Lett 1997; 410:78-82; PMID:9247127; http://dx.doi.org/10.1016/S0014-5793(97)00323-2

31. De Palma C, Morisi F, Cheli S, Pambianco S, Cappello V, Vezzoli M, Rovere-Querini P, Moggio M, Ripolone M, Francolini M, et al. Autophagy as a new therapeutic target in Duchenne muscular dystrophy. Cell Death Dis 2012; 3:e418; PMID:23152054; http://dx.doi.org/10.1038/cddis.2012.159

32. Grumati P, Coletto L, Sabatelli P, Cescon M, Angelin A, Bertaggia E, Blaauw B, Urciuolo A, Tiepolo T, Merlini L, et al. Autophagy is defective in collagen VI muscular dystrophies, and its reactivation rescues myofiber degeneration. Nat Med 2010; 16:1313-20; PMID:21037586; http://dx.doi.org/10.1038/nm.2247

33. Coffer PJ, Jin J, Woodgett JR. Protein kinase B (c-Akt): a multifunctional mediator of phosphatidylinositol 3-kinase activation. Biochem J 1998; 335:1-13; PMID:9742206

34. Paul PK, Bhatnagar S, Mishra V, Srivastava S, Darnay BG, Choi Y, Kumar A. The E3 ubiquitin ligase TRAF6 intercedes in starvation-induced skeletal muscle atrophy through multiple mechanisms. Mol Cell Biol 2012; 32:1248-59; PMID:22290431; http://dx.doi.org/10.1128/MCB.06351-11

35. Pardo PS, Lopez MA, Boriek AM. FOXO transcription factors are mechanosensitive and their regulation is altered with aging in the respiratory pump. Am J Physiol Cell Physiol 2008; 294:C1056-66; PMID:18272820; http://dx.doi.org/10.1152/ajpcell.00270.2007

36. Hosokawa N, Hara T, Kaizuka T, Kishi C, Takamura A, Miura Y, Iemura S, Natsume T, Takehana K, Yamada N, et al. Nutrient-dependent mTORC1 association with the ULK1-Atg13-FIP200 complex required for autophagy. Mol Biol Cell 2009; 20:1981-91; PMID:19211835; http://dx.doi.org/10.1091/mbc.E08-12-1248

37. Jung CH, Jun CB, Ro SH, Kim YM, Otto NM, Cao J, Kundu M, Kim DH. ULK-Atg13-FIP200 complexes mediate mTOR signaling to the autophagy machinery. Mol Biol Cell 2009; 20:1992-2003; PMID:19225151; http://dx.doi.org/10.1091/mbc.E08-12-1249

38. Ganley IG, Lam H, Wang J, Ding X, Chen S, Jiang X. ULK1.ATG13.FIP200 complex mediates mTOR signaling and is essential for autophagy. J Biol Chem 2009; 284:12297-305; PMID:19258318; http://dx.doi.org/10.1074/jbc.M900573200

39. Shang L, Chen S, Du F, Li S, Zhao L, Wang X. Nutrient starvation elicits an acute autophagic response mediated by Ulk1 dephosphorylation and its subsequent dissociation from AMPK. Proc Natl Acad Sci U S A 2011; 108:4788-93; PMID:21383122; http://dx.doi.org/10.1073/pnas.1100844108

40. Lee JW, Park S, Takahashi Y, Wang HG. The association of AMPK with ULK1 regulates autophagy. PLoS One 2010; 5:e15394; PMID:21072212; http://dx.doi.org/10.1371/journal.pone.0015394

41. Djavaheri-Mergny M, Amelotti M, Mathieu J, Besançon F, Bauvy C, Souquère S, Pierron G, Codogno P. NF-kappaB activation represses tumor necrosis factor-alpha-induced autophagy. J Biol Chem 2006; 281:30373-82; PMID:16857678; http://dx.doi.org/10.1074/jbc.M602097200

42. Chen Y, Azad MB, Gibson SB. Superoxide is the major reactive oxygen species regulating autophagy. Cell Death Differ 2009; 16:1040-52; PMID:19407826; http://dx.doi.org/10.1038/cdd.2009.49

43. Kirkland RA, Saavedra GM, Franklin JL. Rapid activation of antioxidant defenses by nerve growth factor suppresses reactive oxygen species during neuronal apoptosis: evidence for a role in cytochrome c redistribution. J Neurosci 2007; 27:11315-26; PMID:17942726; http://dx.doi.org/10.1523/JNEUROSCI.3590-07.2007

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=16125114&dopt=Abstract


http://dx.doi.org/10.1016/j.biocel.2004.12.012

http://dx.doi.org/10.1016/j.biocel.2004.12.012



http://dx.doi.org/10.1152/japplphysiol.01288.2005



http://dx.doi.org/10.1164/ajrccm/148.2.281

http://dx.doi.org/10.1164/ajrccm/148.2.281


http://dx.doi.org/10.1016/j.febslet.2010.01.056

http://dx.doi.org/10.1016/j.febslet.2010.01.056


http://dx.doi.org/10.1091/mbc.E03-09-0704



http://dx.doi.org/10.1016/j.cmet.2007.11.001






http://dx.doi.org/10.1073/pnas.1121159109







http://dx.doi.org/10.1016/j.bbrc.2010.02.130

http://dx.doi.org/10.1016/j.bbrc.2010.02.130


http://dx.doi.org/10.1089/ars.2010.3799

http://dx.doi.org/10.1089/ars.2010.3799


http://dx.doi.org/10.1016/j.tcb.2007.07.009

http://dx.doi.org/10.1016/j.tcb.2007.07.009


http://dx.doi.org/10.1038/emboj.2010.60

http://dx.doi.org/10.1038/emboj.2010.60


http://dx.doi.org/10.1111/j.1748-1716.2010.02226.x

http://dx.doi.org/10.1111/j.1748-1716.2010.02226.x


http://dx.doi.org/10.1113/jphysiol.2012.230185


http://dx.doi.org/10.4161/auto.7.6.15100




http://dx.doi.org/10.1126/science.1196371

http://dx.doi.org/10.1126/science.1196371



http://dx.doi.org/10.1038/ncb2152


http://dx.doi.org/10.1128/MCB.06159-11



http://dx.doi.org/10.1158/0008-5472.CAN-05-1134

http://dx.doi.org/10.1158/0008-5472.CAN-05-1134


http://dx.doi.org/10.1042/BST0340722

http://dx.doi.org/10.1042/BST0340722




http://dx.doi.org/10.1016/j.celrep.2012.03.014

http://dx.doi.org/10.1016/j.celrep.2012.03.014





http://dx.doi.org/10.1016/j.molcel.2012.05.040

http://dx.doi.org/10.1016/j.molcel.2012.05.040


http://dx.doi.org/10.1074/jbc.271.44.27879

http://dx.doi.org/10.1074/jbc.271.44.27879


http://dx.doi.org/10.1016/S0014-5793(97)00323-2

http://dx.doi.org/10.1016/S0014-5793(97)00323-2


http://dx.doi.org/10.1038/cddis.2012.159


http://dx.doi.org/10.1038/nm.2247

http://dx.doi.org/10.1038/nm.2247





http://dx.doi.org/10.1152/ajpcell.00270.2007

http://dx.doi.org/10.1152/ajpcell.00270.2007









http://dx.doi.org/10.1074/jbc.M900573200



http://dx.doi.org/10.1073/pnas.1100844108


http://dx.doi.org/10.1371/journal.pone.0015394

http://dx.doi.org/10.1371/journal.pone.0015394





http://dx.doi.org/10.1038/cdd.2009.49



http://dx.doi.org/10.1523/JNEUROSCI.3590-07.2007

http://dx.doi.org/10.1523/JNEUROSCI.3590-07.2007

https://www.researchgate.net/publication/50989510_Characterization_of_macroautophagic_flux_in_vivo_using_a_leupeptin-based_assay?el=1_x_8&enrichId=rgreq-65d7987d-f425-42f1-86c0-159214d7a7af&enrichSource=Y292ZXJQYWdlOzI1NTk3NTA3NjtBUzoxMDM4MjQ3NTI2NDQxMDdAMTQwMTc2NTE1ODc3OA==





https://www.researchgate.net/publication/220000787_The_E3_ubiquitin_ligase_TRAF6_intercedes_starvation-induced_skeletal_muscle_atrophy_through_multiple_mechanisms?el=1_x_8&enrichId=rgreq-65d7987d-f425-42f1-86c0-159214d7a7af&enrichSource=Y292ZXJQYWdlOzI1NTk3NTA3NjtBUzoxMDM4MjQ3NTI2NDQxMDdAMTQwMTc2NTE1ODc3OA==






https://www.researchgate.net/publication/14297471_Characterization_of_the_AMP-activated_Protein_Kinase_Kinase_from_Rat_Liver_and_Identification_of_Threonine_172_as_the_Major_Site_at_Which_It_Phosphorylates_AMP-activated_Protein_Kinase?el=1_x_8&enrichId=rgreq-65d7987d-f425-42f1-86c0-159214d7a7af&enrichSource=Y292ZXJQYWdlOzI1NTk3NTA3NjtBUzoxMDM4MjQ3NTI2NDQxMDdAMTQwMTc2NTE1ODc3OA==








https://www.researchgate.net/publication/50304265_Nutrient_starvation_elicits_an_acute_autophagic_response_mediated_by_Ulk1_dephosphorylation_and_its_subsequent_dissociation_from_AMPK_Proc_Natl_Acad_Sci_USA?el=1_x_8&enrichId=rgreq-65d7987d-f425-42f1-86c0-159214d7a7af&enrichSource=Y292ZXJQYWdlOzI1NTk3NTA3NjtBUzoxMDM4MjQ3NTI2NDQxMDdAMTQwMTc2NTE1ODc3OA==






https://www.researchgate.net/publication/24394719_Superoxide_is_the_major_reactive_oxygen_species_regulating_autophagy?el=1_x_8&enrichId=rgreq-65d7987d-f425-42f1-86c0-159214d7a7af&enrichSource=Y292ZXJQYWdlOzI1NTk3NTA3NjtBUzoxMDM4MjQ3NTI2NDQxMDdAMTQwMTc2NTE1ODc3OA==




https://www.researchgate.net/publication/51743756_Role_of_AMPK-mTOR-Ulk12_in_the_Regulation_of_Autophagy_Cross_Talk_Shortcuts_and_Feedbacks?el=1_x_8&enrichId=rgreq-65d7987d-f425-42f1-86c0-159214d7a7af&enrichSource=Y292ZXJQYWdlOzI1NTk3NTA3NjtBUzoxMDM4MjQ3NTI2NDQxMDdAMTQwMTc2NTE1ODc3OA==





https://www.researchgate.net/publication/5794437_FoxO3_Controls_Autophagy_in_Skeletal_Muscle_In_Vivo?el=1_x_8&enrichId=rgreq-65d7987d-f425-42f1-86c0-159214d7a7af&enrichSource=Y292ZXJQYWdlOzI1NTk3NTA3NjtBUzoxMDM4MjQ3NTI2NDQxMDdAMTQwMTc2NTE1ODc3OA==






https://www.researchgate.net/publication/228866300_Skeletal_muscle_fiber_types_in_C57BL6J_mice?el=1_x_8&enrichId=rgreq-65d7987d-f425-42f1-86c0-159214d7a7af&enrichSource=Y292ZXJQYWdlOzI1NTk3NTA3NjtBUzoxMDM4MjQ3NTI2NDQxMDdAMTQwMTc2NTE1ODc3OA==



https://www.researchgate.net/publication/13973069_The_modular_phosphorylation_and_activation_of_p70S6K?el=1_x_8&enrichId=rgreq-65d7987d-f425-42f1-86c0-159214d7a7af&enrichSource=Y292ZXJQYWdlOzI1NTk3NTA3NjtBUzoxMDM4MjQ3NTI2NDQxMDdAMTQwMTc2NTE1ODc3OA==




https://www.researchgate.net/publication/49777566_AMPK_and_mTOR_regulate_autophagy_via_direct_phosphorylation_of_ULK1?el=1_x_8&enrichId=rgreq-65d7987d-f425-42f1-86c0-159214d7a7af&enrichSource=Y292ZXJQYWdlOzI1NTk3NTA3NjtBUzoxMDM4MjQ3NTI2NDQxMDdAMTQwMTc2NTE1ODc3OA==




https://www.researchgate.net/publication/43183479_Mitochondrial_fission_and_remodelling_contributes_to_muscle_atrophy?el=1_x_8&enrichId=rgreq-65d7987d-f425-42f1-86c0-159214d7a7af&enrichSource=Y292ZXJQYWdlOzI1NTk3NTA3NjtBUzoxMDM4MjQ3NTI2NDQxMDdAMTQwMTc2NTE1ODc3OA==






https://www.researchgate.net/publication/44902036_Myostatin_expression_is_increased_by_food_deprivation_in_a_muscle-specific_manner_and_contributes_to_muscle_atrophy_during_prolonged_food_deprivation_in_mice?el=1_x_8&enrichId=rgreq-65d7987d-f425-42f1-86c0-159214d7a7af&enrichSource=Y292ZXJQYWdlOzI1NTk3NTA3NjtBUzoxMDM4MjQ3NTI2NDQxMDdAMTQwMTc2NTE1ODc3OA==






https://www.researchgate.net/publication/23559522_Skeletal_Muscle_Is_a_Primary_Target_of_SOD1G93A-Mediated_Toxicity?el=1_x_8&enrichId=rgreq-65d7987d-f425-42f1-86c0-159214d7a7af&enrichSource=Y292ZXJQYWdlOzI1NTk3NTA3NjtBUzoxMDM4MjQ3NTI2NDQxMDdAMTQwMTc2NTE1ODc3OA==






https://www.researchgate.net/publication/5794438_FoxO3_Coordinately_Activates_Protein_Degradation_by_the_AutophagicLysosomal_and_Proteasomal_Pathways_in_Atrophying_Muscle_Cells?el=1_x_8&enrichId=rgreq-65d7987d-f425-42f1-86c0-159214d7a7af&enrichSource=Y292ZXJQYWdlOzI1NTk3NTA3NjtBUzoxMDM4MjQ3NTI2NDQxMDdAMTQwMTc2NTE1ODc3OA==







https://www.researchgate.net/publication/233418681_Autophagy_as_a_new_therapeutic_target_in_Duchenne_muscular_dystrophy?el=1_x_8&enrichId=rgreq-65d7987d-f425-42f1-86c0-159214d7a7af&enrichSource=Y292ZXJQYWdlOzI1NTk3NTA3NjtBUzoxMDM4MjQ3NTI2NDQxMDdAMTQwMTc2NTE1ODc3OA==






https://www.researchgate.net/publication/228089751_Mouse_Skeletal_Muscle_Fiber-Type-Specific_Macroautophagy_and_Muscle_Wasting_Are_Regulated_by_a_FynSTAT3Vps34_Signaling_Pathway?el=1_x_8&enrichId=rgreq-65d7987d-f425-42f1-86c0-159214d7a7af&enrichSource=Y292ZXJQYWdlOzI1NTk3NTA3NjtBUzoxMDM4MjQ3NTI2NDQxMDdAMTQwMTc2NTE1ODc3OA==






https://www.researchgate.net/publication/6912557_Lysosomal_Turnover_but_Not_a_Cellular_Level_of_Endogenous_LC3_is_a_Marker_for_Autophagy?el=1_x_8&enrichId=rgreq-65d7987d-f425-42f1-86c0-159214d7a7af&enrichSource=Y292ZXJQYWdlOzI1NTk3NTA3NjtBUzoxMDM4MjQ3NTI2NDQxMDdAMTQwMTc2NTE1ODc3OA==





https://www.researchgate.net/publication/24023143_ULK-Atg13-FIP200_complexes_mediate_mTOR_signaling_to_the_autophagy_machinery_Mol_Biol_Cell?el=1_x_8&enrichId=rgreq-65d7987d-f425-42f1-86c0-159214d7a7af&enrichSource=Y292ZXJQYWdlOzI1NTk3NTA3NjtBUzoxMDM4MjQ3NTI2NDQxMDdAMTQwMTc2NTE1ODc3OA==






https://www.researchgate.net/publication/47636282_Grumati_P_Coletto_L_Sabatelli_P_et_alAutophagy_is_defective_in_collagen_VI_muscular_dystrophies_and_its_reactivation_rescues_myofiber_degeneration_Nat_Med_161313-1320?el=1_x_8&enrichId=rgreq-65d7987d-f425-42f1-86c0-159214d7a7af&enrichSource=Y292ZXJQYWdlOzI1NTk3NTA3NjtBUzoxMDM4MjQ3NTI2NDQxMDdAMTQwMTc2NTE1ODc3OA==







https://www.researchgate.net/publication/8939375_In_Vivo_Analysis_of_Autophagy_in_Response_to_Nutrient_Starvation_Using_Transgenic_Mice_Expressing_a_Fluorescent_Autophagosome_Marker?el=1_x_8&enrichId=rgreq-65d7987d-f425-42f1-86c0-159214d7a7af&enrichSource=Y292ZXJQYWdlOzI1NTk3NTA3NjtBUzoxMDM4MjQ3NTI2NDQxMDdAMTQwMTc2NTE1ODc3OA==






https://www.researchgate.net/publication/24002924_Nutrient-dependent_mTORC1_association_with_the_ULK1-Atg13-FIP200_complex_required_for_autophagy_Mol_Biol_Cell?el=1_x_8&enrichId=rgreq-65d7987d-f425-42f1-86c0-159214d7a7af&enrichSource=Y292ZXJQYWdlOzI1NTk3NTA3NjtBUzoxMDM4MjQ3NTI2NDQxMDdAMTQwMTc2NTE1ODc3OA==







https://www.researchgate.net/publication/228086934_Bim_Inhibits_Autophagy_by_Recruiting_Beclin_1_to_Microtubules?el=1_x_8&enrichId=rgreq-65d7987d-f425-42f1-86c0-159214d7a7af&enrichSource=Y292ZXJQYWdlOzI1NTk3NTA3NjtBUzoxMDM4MjQ3NTI2NDQxMDdAMTQwMTc2NTE1ODc3OA==





https://www.researchgate.net/publication/7635882_Calpains_in_muscle_wasting?el=1_x_8&enrichId=rgreq-65d7987d-f425-42f1-86c0-159214d7a7af&enrichSource=Y292ZXJQYWdlOzI1NTk3NTA3NjtBUzoxMDM4MjQ3NTI2NDQxMDdAMTQwMTc2NTE1ODc3OA==





https://www.researchgate.net/publication/5578938_FOXO_transcription_factors_are_mechanosensitive_and_their_regulation_is_altered_with_aging_in_the_respiratory_pump?el=1_x_8&enrichId=rgreq-65d7987d-f425-42f1-86c0-159214d7a7af&enrichSource=Y292ZXJQYWdlOzI1NTk3NTA3NjtBUzoxMDM4MjQ3NTI2NDQxMDdAMTQwMTc2NTE1ODc3OA==






https://www.researchgate.net/publication/41407671_Autophagy_in_skeletal_muscle?el=1_x_8&enrichId=rgreq-65d7987d-f425-42f1-86c0-159214d7a7af&enrichSource=Y292ZXJQYWdlOzI1NTk3NTA3NjtBUzoxMDM4MjQ3NTI2NDQxMDdAMTQwMTc2NTE1ODc3OA==



https://www.researchgate.net/publication/45288070_Quantitation_of_autophagic_flux_in_mature_skeletal_muscle?el=1_x_8&enrichId=rgreq-65d7987d-f425-42f1-86c0-159214d7a7af&enrichSource=Y292ZXJQYWdlOzI1NTk3NTA3NjtBUzoxMDM4MjQ3NTI2NDQxMDdAMTQwMTc2NTE1ODc3OA==




https://www.researchgate.net/publication/7597440_Blockade_of_Epidermal_Growth_Factor_Receptors_Chemosensitizes_Breast_Cancer_Cells_through_Up-Regulation_of_Bnip3L?el=1_x_8&enrichId=rgreq-65d7987d-f425-42f1-86c0-159214d7a7af&enrichSource=Y292ZXJQYWdlOzI1NTk3NTA3NjtBUzoxMDM4MjQ3NTI2NDQxMDdAMTQwMTc2NTE1ODc3OA==







https://www.researchgate.net/publication/7293463_Caspase_activation_contributes_to_endotoxin-induced_diaphragm_weakness?el=1_x_8&enrichId=rgreq-65d7987d-f425-42f1-86c0-159214d7a7af&enrichSource=Y292ZXJQYWdlOzI1NTk3NTA3NjtBUzoxMDM4MjQ3NTI2NDQxMDdAMTQwMTc2NTE1ODc3OA==






https://www.researchgate.net/publication/47789101_Effect_of_LKB1_deficiency_on_mitochondrial_content_fibre_type_and_muscle_performance_in_the_mouse_diaphragm?el=1_x_8&enrichId=rgreq-65d7987d-f425-42f1-86c0-159214d7a7af&enrichSource=Y292ZXJQYWdlOzI1NTk3NTA3NjtBUzoxMDM4MjQ3NTI2NDQxMDdAMTQwMTc2NTE1ODc3OA==







https://www.researchgate.net/publication/224958861_Biomarkers_of_mitochondrial_content_in_skeletal_muscle_of_healthy_young_human_subjects?el=1_x_8&enrichId=rgreq-65d7987d-f425-42f1-86c0-159214d7a7af&enrichSource=Y292ZXJQYWdlOzI1NTk3NTA3NjtBUzoxMDM4MjQ3NTI2NDQxMDdAMTQwMTc2NTE1ODc3OA==






https://www.researchgate.net/publication/221808145_Histone_deacetylases_1_and_2_regulate_autophagy_flux_and_skeletal_muscle_homeostasis_in_mice?el=1_x_8&enrichId=rgreq-65d7987d-f425-42f1-86c0-159214d7a7af&enrichSource=Y292ZXJQYWdlOzI1NTk3NTA3NjtBUzoxMDM4MjQ3NTI2NDQxMDdAMTQwMTc2NTE1ODc3OA==




https://www.researchgate.net/publication/6036703_ROS_mitochondria_and_the_regulation_of_autophagy?el=1_x_8&enrichId=rgreq-65d7987d-f425-42f1-86c0-159214d7a7af&enrichSource=Y292ZXJQYWdlOzI1NTk3NTA3NjtBUzoxMDM4MjQ3NTI2NDQxMDdAMTQwMTc2NTE1ODc3OA==




https://www.researchgate.net/publication/40038545_Autophagy_Is_Required_to_Maintain_Muscle_Mass?el=1_x_8&enrichId=rgreq-65d7987d-f425-42f1-86c0-159214d7a7af&enrichSource=Y292ZXJQYWdlOzI1NTk3NTA3NjtBUzoxMDM4MjQ3NTI2NDQxMDdAMTQwMTc2NTE1ODc3OA==





https://www.researchgate.net/publication/41577565_Fasting-related_autophagic_response_in_slow-_and_fast-twitch_skeletal_muscle?el=1_x_8&enrichId=rgreq-65d7987d-f425-42f1-86c0-159214d7a7af&enrichSource=Y292ZXJQYWdlOzI1NTk3NTA3NjtBUzoxMDM4MjQ3NTI2NDQxMDdAMTQwMTc2NTE1ODc3OA==





https://www.researchgate.net/publication/49717484_Targeting_Mitochondrial_Dysfunction_Role_for_PINK1_and_Parkin_in_Mitochondrial_Quality_Control?el=1_x_8&enrichId=rgreq-65d7987d-f425-42f1-86c0-159214d7a7af&enrichSource=Y292ZXJQYWdlOzI1NTk3NTA3NjtBUzoxMDM4MjQ3NTI2NDQxMDdAMTQwMTc2NTE1ODc3OA==





https://www.researchgate.net/publication/6743984_FOXO_transcription_factors_Key_regulators_of_cell_fate?el=1_x_8&enrichId=rgreq-65d7987d-f425-42f1-86c0-159214d7a7af&enrichSource=Y292ZXJQYWdlOzI1NTk3NTA3NjtBUzoxMDM4MjQ3NTI2NDQxMDdAMTQwMTc2NTE1ODc3OA==




https://www.researchgate.net/publication/47756116_The_association_of_AMPK_with_ULK1_regulates_autophagy?el=1_x_8&enrichId=rgreq-65d7987d-f425-42f1-86c0-159214d7a7af&enrichSource=Y292ZXJQYWdlOzI1NTk3NTA3NjtBUzoxMDM4MjQ3NTI2NDQxMDdAMTQwMTc2NTE1ODc3OA==




https://www.researchgate.net/publication/281949542_NF-kappaB_activation_represses_tumor_necrosis_factor-alpha-induced_autophagy?el=1_x_8&enrichId=rgreq-65d7987d-f425-42f1-86c0-159214d7a7af&enrichSource=Y292ZXJQYWdlOzI1NTk3NTA3NjtBUzoxMDM4MjQ3NTI2NDQxMDdAMTQwMTc2NTE1ODc3OA==






https://www.researchgate.net/publication/14856190_Tumor_Necrosis_Factor_and_Endotoxin_Do_Not_Directly_Affect_In_Vitro_Diaphragm_Function?el=1_x_8&enrichId=rgreq-65d7987d-f425-42f1-86c0-159214d7a7af&enrichSource=Y292ZXJQYWdlOzI1NTk3NTA3NjtBUzoxMDM4MjQ3NTI2NDQxMDdAMTQwMTc2NTE1ODc3OA==





©20

13 L

ande

s B

iosc

ienc

e. D

o no

t dis

tribu

te.


44. Scherz-Shouval R, Elazar Z. Regulation of autophagy by ROS: physiology and pathology. Trends Biochem Sci 2011; 36:30-8; PMID:20728362; http://dx.doi.org/10.1016/j.tibs.2010.07.007

45. Narendra D, Tanaka A, Suen DF, Youle RJ. Parkin-induced mitophagy in the pathogenesis of Parkinson disease. Autophagy 2009; 5:706-8; PMID:19377297; http://dx.doi.org/10.4161/auto.5.5.8505

46. Yeo JE, Kim JH, Kang SK. Selenium attenuates ROS-mediated apoptotic cell death of injured spinal cord through prevention of mitochondria dysfunction; in vitro and in vivo study. Cell Physiol Biochem 2008; 21:225-38; PMID:18209489; http://dx.doi.org/10.1159/000113764

47. Davis EC. Smooth muscle cell to elastic lamina connections in developing mouse aorta. Role in aortic medial organization. Lab Invest 1993; 68:89-99; PMID:8423679

48. Mofarrahi M, Nouh T, Qureshi S, Guillot L, Mayaki D, Hussain SNA. Regulation of angiopoietin expression by bacterial lipopolysaccharide. Am J Physiol Lung Cell Mol Physiol 2008; 294:L955-63; PMID:18310225; http://dx.doi.org/10.1152/ajplung.00449.2007

49. Picard M, Ritchie D, Wright KJ, Romestaing C, Thomas MM, Rowan SL, Taivassalo T, Hepple RT. Mitochondrial functional impairment with aging is exaggerated in isolated mitochondria compared to permeabilized myofibers. Aging Cell 2010; 9:1032-46; PMID:20849523; http://dx.doi.org/10.1111/j.1474-9726.2010.00628.x


http://dx.doi.org/10.1016/j.tibs.2010.07.007

http://dx.doi.org/10.1016/j.tibs.2010.07.007




http://dx.doi.org/10.1159/000113764

http://dx.doi.org/10.1159/000113764




http://dx.doi.org/10.1152/ajplung.00449.2007

http://dx.doi.org/10.1152/ajplung.00449.2007


http://dx.doi.org/10.1111/j.1474-9726.2010.00628.x

http://dx.doi.org/10.1111/j.1474-9726.2010.00628.x

https://www.researchgate.net/publication/5542626_Regulation_of_angiopoietin_expression_by_bacterial_lipopolysaccharide?el=1_x_8&enrichId=rgreq-65d7987d-f425-42f1-86c0-159214d7a7af&enrichSource=Y292ZXJQYWdlOzI1NTk3NTA3NjtBUzoxMDM4MjQ3NTI2NDQxMDdAMTQwMTc2NTE1ODc3OA==






https://www.researchgate.net/publication/24308264_Parkin-induced_mitophagy_in_the_pathogenesis_of_Parkinson_disease_Autophagy_5706-708?el=1_x_8&enrichId=rgreq-65d7987d-f425-42f1-86c0-159214d7a7af&enrichSource=Y292ZXJQYWdlOzI1NTk3NTA3NjtBUzoxMDM4MjQ3NTI2NDQxMDdAMTQwMTc2NTE1ODc3OA==




https://www.researchgate.net/publication/46303761_Mitochondrial_functional_impairment_with_aging_is_exaggerated_in_isolated_mitochondria_compared_to_permeabilized_myofibers?el=1_x_8&enrichId=rgreq-65d7987d-f425-42f1-86c0-159214d7a7af&enrichSource=Y292ZXJQYWdlOzI1NTk3NTA3NjtBUzoxMDM4MjQ3NTI2NDQxMDdAMTQwMTc2NTE1ODc3OA==







https://www.researchgate.net/publication/45798831_Regulation_of_autophagy_by_ROS_physiology_and_pathology_Trends_Biochem_Sci?el=1_x_8&enrichId=rgreq-65d7987d-f425-42f1-86c0-159214d7a7af&enrichSource=Y292ZXJQYWdlOzI1NTk3NTA3NjtBUzoxMDM4MjQ3NTI2NDQxMDdAMTQwMTc2NTE1ODc3OA==




https://www.researchgate.net/publication/5643203_Selenium_Attenuates_ROS-Mediated_Apoptotic_Cell_Death_of_Injured_Spinal_Cord_through_Prevention_of_Mitochondria_Dysfunction_in_Vitro_and_in_Vivo_Study?el=1_x_8&enrichId=rgreq-65d7987d-f425-42f1-86c0-159214d7a7af&enrichSource=Y292ZXJQYWdlOzI1NTk3NTA3NjtBUzoxMDM4MjQ3NTI2NDQxMDdAMTQwMTc2NTE1ODc3OA==






https://www.researchgate.net/publication/14774989_Smooth_muscle_cell_to_elastic_lamina_connections_in_developing_mouse_aorta_Role_in_aortic_medial_organization?el=1_x_8&enrichId=rgreq-65d7987d-f425-42f1-86c0-159214d7a7af&enrichSource=Y292ZXJQYWdlOzI1NTk3NTA3NjtBUzoxMDM4MjQ3NTI2NDQxMDdAMTQwMTc2NTE1ODc3OA==




Autophagic flux and oxidative capacity of skeletal muscles during acute starvation

Documents

Transcript of Autophagic flux and oxidative capacity of skeletal muscles during acute starvation