Modulation of autophagy signaling with resistance exercise and protein ingestion following short-term energy deficit
Abstract
Autophagy contributes to remodeling of skeletal muscle and is sensitive to contractile activity and prevailing energy availability. We investigated changes in targeted genes and proteins with roles in autophagy following 5 days of energy balance (EB), energy deficit (ED), and resistance exercise (REX) after ED. Muscle biopsies from 15 subjects (8 males, 7 females) were taken at rest following 5 days of EB [45 kcal·kg fat free mass (FFM)−1·day−1] and 5 days of ED (30 kcal·kg FFM−1·day−1). After ED, subjects completed a bout of REX and consumed either placebo (PLA) or 30 g whey protein (PRO) immediately postexercise. Muscle biopsies were obtained at 1 and 4 h into recovery in each trial. Resting protein levels of autophagy-related gene protein 5 (Atg5) decreased after ED compared with EB (∼23%, P < 0.001) and remained below EB from 1 to 4 h postexercise in PLA (∼17%) and at 1 h in PRO (∼18%, P < 0.05). In addition, conjugated Atg5 (cAtg12) decreased below EB in PLA at 4 h (∼20, P < 0.05); however, its values were increased above this time point in PRO at 4 h alongside increases in FOXO1 above EB (∼22–26%, P < 0.05). Notably, these changes were subsequent to increases in unc-51-like kinase 1Ser757 phosphorylation (∼60%) 1 h postexercise in PRO. No significant changes in gene expression of selected autophagy markers were found, but EGR-1 increased above ED and EB in PLA (∼417–864%) and PRO (∼1,417–2,731%) trials 1 h postexercise (P < 0.001). Postexercise protein availability, compared with placebo, can selectively promote autophagic responses to REX in ED.
maintenance and remodeling of skeletal muscle mass depends on the net balance between simultaneous processes of muscle protein synthesis (MPS) and muscle protein breakdown (MPB). Periods of energy deficit (ED) are associated with losses of lean body mass (35), and we (2) and others (44) have demonstrated that short-term ED through dietary restriction reduces postabsorptive rates of MPS compared with energy balance (EB). The protein degradation signaling pathways underlying this increased MPB response are not well characterized, although increased mRNA expression of ubiquitin proteasomal system (UPS) markers have been reported following short-term ED (9, 10). However, this UPS response is repressed following protein consumption (9), suggesting a possible muscle protein “sparing” effect of protein ingestion when in ED.
The loss of muscle proteins resulting from ED (35) may, in part, be attributed to enhanced activity of the autophagy-lysosomal pathway (henceforth, termed autophagy). For example, chronic (6 mo) ED increases mRNA and protein markers of autophagy (53). Autophagy degrades, via lysosomal proteolysis, protein aggregates and damaged organelles that are then “recycled” to yield energy to maintain cellular metabolism (11). This process commences with the sequestration of a portion of cytoplasm by a double-membrane vacuole called the autophagosome, which subsequently releases its contents for degradation upon fusion with the lysosome (37). Formation, expansion, and lysosomal fusion of the autophagosome is coordinated by subgroups of autophagy-related gene (Atg) proteins, collectively referred to as the core molecular machinery (55).
Increases in protein levels and mRNA transcripts of Atgs observed following endurance exercise have been proposed to “clear” accumulative cell debris and alleviate metabolic perturbations induced by contractile activity (21, 22, 27, 38, 48). This autophagic response may be the result of attenuated insulin/Akt signaling and intracellular energy that promotes autophagy through activation of upstream energy- and stress-responsive kinases. Autophagy conceivably initiates an adaptive response to this contractile stimulus by restoring EB and facilitating an ultimate transition to intracellular anabolism through protein and nutrient resynthesis. In contrast to endurance exercise, several markers of autophagy decrease or remain unchanged following resistance exercise (REX) in humans (14, 15). This highlights ubiquitin-mediated proteolysis as the predominant degradation pathway responsible for initial increases in MPB following REX (45) and may indicate that augmenting the activation of autophagy is dependent on a substantial cellular energy perturbation. Whether the combined effects of short-term ED and REX activate muscle autophagy is unknown. Moreover, the effects of protein ingestion on autophagy signaling following ED and REX have not been determined. Amino acids have been shown to regulate autophagic flux (40), but these effects are largely unknown in human skeletal muscle. Accordingly, the aim of this study was to investigate targeted autophagy signaling responses at rest following 5 days ED and subsequently after REX and protein ingestion undertaken in ED. We hypothesized that ED would increase autophagy signaling, and this response would be exacerbated by the metabolic stress induced by REX. We also hypothesized that increasing protein availability after REX would reduce the magnitude of autophagy signaling.
MATERIALS AND METHODS
Subjects
Fifteen (8 males, 7 females) young, healthy, resistance-trained subjects [male: age 27 ± 5 yr, body mass (BM) 82.7 ± 6.6 kg, leg press 1-repetition maximum (1-RM) 300 ± 70 kg; female: age 28 ± 4 yr, BM 70.3 ± 7 kg, 1-RM 200 ± 28 kg; values are means ± SD] were recruited in a previous study (2). Subjects were advised of any possible risks associated with the study prior to providing written informed consent. The study was approved by the Australian Institute of Sport Ethics Committee and conformed to the standards set by the latest revision of the Declaration of Helsinki.
Experimental Design
The study employed a within-subject design in which each subject completed four experimental interventions for measures of resting energy balance (EB), resting ED, and then REX following ED with ingestion of protein or a nonnutrient placebo (PRO and PLA trials, respectively). The EB trial was always undertaken before ED trials to eliminate any possible metabolic disruptions elicited by a previous ED intervention (Fig. 1). For ED trials, the protein/placebo ingestion following REX was randomized and counterbalanced.
Fig. 1.Overview of experimental design. The resting energy balance (EB) trial was preceded by 5 days of a controlled diet that provided 45 kcal·kg FFM−1·day−1. The energy deficit (ED) trials were preceded by 5 days of a controlled diet providing 30 kcal·kg FFM−1·day−1. Solid black arrow denotes muscle biopsy sample. PLA and PRO represent the respective placebo or 30 g whey protein drink (500 ml). Dashed timeline (EB trial) represent trials undertaken a single time by each subject. Times in parentheses are for ED trials involving exercise and beverage intake.
Maximal Strength
Lower body strength was determined 1 wk prior to experimental trials during a series of single repetitions on an inclined (45°) leg press (GLPH1100, Body-Solid, Forest Park IL) until the maximal load was established (1-RM).
Dietary Intervention
As previously published (2), subjects received individualized prepackaged meals for 5 days prior to an experimental trial. Before the resting EB trial, subjects received meals equivalent to an energy availability of 45 kcal·kg fat-free mass (FFM)−1·day−1, where energy availability was defined as energy intake minus the energy cost of habitual exercise. For ED trials, diets consisted of an energy availability of 30 kcal·kg FFM−1·day−1. From days 1–3 of the dietary control period, subjects were permitted to exercise, and the diet was adjusted to account for the energy expenditure of exercise sessions to maintain energy availability at a set level. In the 48 h prior to an experimental trial (days 4 and 5 of dietary control), subjects refrained from any strenuous physical activity. No alcohol was consumed during the 5-day dietary control period, and subjects refrained from caffeine intake 24 h before each trial day. The 5-day ED period was based on previous studies showing that this timeframe is sufficient to cause perturbations in whole-body metabolic homeostasis (20, 30). The protein, carbohydrate, and fat content of the diets was 1.4–1.6, 4–4.5, and 1.5–2.5 g·kg body mass (BM)−1·day−1 for EB and 1.4–1.6, 3–3.5, and 0.5–1.5 g·kg BM−1·day−1 for ED, respectively (2). Experimental trials were separated by a 9-day washout period based on data demonstrating that the reduction in resting metabolic rate after a 20-day ED can be restored with 10 days of EB (51). During the washout period, subjects resumed their habitual exercise and diet routine.
Experimental Trials
Following 5 days of dietary control, subjects reported to the laboratory between 0700 and 0800 after a 10–12-h overnight fast. In both EB and ED trials, subjects rested for a 3-h period, and a muscle biopsy was obtained from the vastus lateralis under local anesthesia (1% lidocaine) using a 5-mm Bergstrom needle modified for suction. For the ED trials, subjects completed a standardized warm-up (2 × 5 repetitions at ∼50 and ∼60% 1-RM) on a leg-press machine before commencing the REX protocol comprising six sets of eight repetitions at ∼80% 1-RM (3-min recovery between sets). In a randomized and counterbalanced design, subjects ingested 500 ml of either placebo (PLA: water, vanilla flavor, artificial sweetener) or a protein beverage (PRO: 30 g, ISO8 vanilla WPI; 86.8 g protein, 1.5 g fat, 3.1 g carbohydrates/100 g; Musashi, Australia) on separate occasions. Subjects would rest throughout a 240-min recovery period during which additional muscle biopsies were obtained at 60 and 240 min postexercise (Fig. 1). All muscle samples were stored at −80°C for subsequent analysis.
Analytical Procedures
Western blot analysis.
Approximately 40 mg of skeletal muscle was homogenized in a buffer containing 50 mM Tris·HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 1% Triton X-100, 50 mM NaF, 5 mM sodium pyrophosphate, 1 mM DTT, 10 μg/ml trypsin inhibitor, 2 μg/ml aprotinin, 1 mM benzamidine, and 1 mM PMSF. Samples were spun at 18,000 g for 30 min at 4°C, and the supernatant was collected for Western blot analysis. After determination of protein concentration using a BCA protein assay (Pierce Biotechnology, Rockford, IL), lysate was resuspended in Laemmli sample buffer; separated by SDS-PAGE; transferred to polyvinylidine fluoride membranes; blocked with 5% nonfat milk; washed with 10 mM Tris·HCl, 100 mM NaCl, and 0.02% Tween 20; and incubated with a primary antibody (1:1,000) overnight at 4°C on a shaker. Membranes were incubated the next day with a secondary antibody (1:2,000), and proteins were detected via enhanced chemiluminescence (Amersham Biosciences, Buckinghamshire, UK; Pierce) and quantified by densitometry (Chemidoc, Bio-Rad, Gladesville, Australia). All sample (40 μg protein) time points for each subject were run singularly on the same gel. Polyclonal antibodies against phosphorylated p38 mitogen-activated protein kinase (p38 MAPK)Thr180/Tyr182 (no. 4511), p53Ser15 (no. 9284), unc-51-like kinase 1 (ULK1)Ser757 (no. 6888), Forkhead box O1 (FOXO1)Thr24 (no. 9464), FOXO3aSer253 (no. 9466), eukaryotic initiation factor 2α (eIF2α)Ser51 (no. 9721), total autophagy-related gene protein 5 (Atg5) (no. 2630), Beclin-1 (no. 3738), microtubule-associated protein-1 light chain 3 beta (LC3b) (no. 2775), cAtg12 (no. 4180), p62 (no. 5114), ULK1 (no. 6439), p53 (no. 2527), p38 MAPK (no. 9212), FOXO1 (no. 9454), FOXO3a (no. 2497), and eIF2α (no. 9722) were all purchased from Cell Signaling Technology (Danvers, MA). The LC3b antibody detects bands for both the 14-kDa LC3b-I and 16 -kDa LC3b-II isoforms. As we were unable to gain consistent separation between bands for the two isoforms, only data for the more prominent LC3b-I band are presented. The presence of this specific band was verified by the use of an LC3 control extract (no. 11972) purchased from Cell Signaling Technology. For all proteins, volume density of each target band was normalized to the total protein loaded into each lane using stain-free technology (18).
RNA extraction and quantification.
Skeletal muscle tissue RNA extraction was performed using a TRIzol-based kit, according to the manufacturer's protocol (Invitrogen, Melbourne, Australia; cat. no. 12183-018A). Briefly, ∼20 mg of skeletal muscle tissue was removed from RNAlater-ICE solution and homogenized in TRIzol. After elution through a spin cartridge, extracted RNA was quantified using a QUANT-iT analyzer kit (Invitrogen, Melbourne, Australia; cat. no. Q32852), according to the manufacturer's protocol.
RT and real-time PCR.
Real-time RT-PCR was performed as previously described (7). Quantification of mRNA (in duplicate) was performed using a Rotor-Gene 3000 centrifugal real-time cycler (Corbett Research, Mortlake, Australia). TaqMan-FAM-labeled primer/probes for LC3b (cat. no. Hs 00797944), γ-aminobutyric-acid-type-A-receptor-associated protein (GABARAP) (cat. no. 00925899), Beclin-1 (cat. no. Hs 00186838), Atg12 (cat. no. Hs 01047860), Atg4b (cat. no. Hs 00367088), sirtuin-1 (SIRT1) (cat. no. Hs 01009005), FOXO1 (cat. no. Hs 01054576), PPARγ-coactivator-1α (PGC-1α) (cat. no. Hs 01016719), Bcl-2/adenovirus E1B 19-kDa interacting protein-3 (BNIP3) (cat. no. Hs 00969291), vascular endothelial growth factor (VEGF) (cat. no. Hs00900055), and early growth response-1 (EGR-1) (cat. no. Hs 00152928) were used. GAPDH (cat. no. Hs 99999905) has been validated as an exercise housekeeping gene (23) and was used to normalize threshold cycle (CT) values. GAPDH values were stably expressed between conditions (data not shown). The relative amount of mRNA was calculated using the relative quantification (ΔΔCT) method (29).
Statistical Analysis
Data were analyzed using two-way repeated-measures ANOVA with Student-Newman-Keuls post hoc analysis (sex × time) (SigmaStat for Windows; version 3.10). As there were no significant differences between sexes for cell signaling and gene expression responses, data were combined for further analysis using a one-way repeated-measures ANOVA (two-tailed) with Student-Newman-Keuls post hoc testing (treatment only). EGR-1 and VEGF mRNA data were log-transformed. Statistical significance was set to P < 0.05, and all data are presented as means ± SD and expressed as arbitrary units.
RESULTS
Cell Signaling Proteins
p38 MAPK-p53-eIF2α.
There were no significant changes in p38 MAPKThr180/Tyr182 phosphorylation at any time point (Fig. 2A); however, total p38 MAPK decreased from EB in PLA at 4 h (∼23%, P < 0.05) and from 1 to 4 h postexercise in PRO (∼24–25%, P < 0.05; Fig. 2B). There were no significant changes in p53Ser15 phosphorylation (Fig. 2C) or total p53 at any time point (Fig. 2D). No significant changes in the phosphorylation state of eIF2αSer51 (Fig. 2E) or the abundance of its corresponding total at any time point were found (Fig. 2F).
Fig. 2.Phospho-p38 MAPKThr180/Tyr182 (A), total p38 MAPK (B) phospho-p53Ser15 (C), total p53 (n = 9; D), phospho-eIF2αSer51 (E), and total eIF2α (F) at rest following 5 days of EB, 5 days of ED, and a bout of leg press (6 sets × 8 repetitions at 80% 1-RM) in ED with postexercise ingestion of a placebo (PLA) or 30 g of a whey protein drink (PRO). Values are expressed relative to total protein loaded determined by stain-free technology and presented in arbitrary units (means ± SD). The letter a denotes significant difference (P < 0.05) vs. EB.
FOXO1, FOXO3a, and LC3b-I.
Although no significant differences in FOXO1Thr24 phosphorylation at any time point were observed (Fig. 3A), there were main treatment effects for total levels of FOXO1 (Fig. 3B). FOXO1 abundance increased in PRO at 4 h above ED and each PLA postexercise time point (∼20–22%, P < 0.05). No significant differences in FOXO3aSer253 phosphorylation (data not shown) or levels of its total were found despite a modest increase in total FOXO3a above resting EB and ED in PLA at 1 h postexercise (Fig. 3C). However, there were main treatment effects for total levels of LC3b-I (Fig. 3D). Total LC3b-I decreased from EB and ED in PLA at 4 h (∼21–26%, P < 0.05) and below EB in PRO at 1 h (∼19%; P = 0.053).
Fig. 3.Phospho-FOXO1Thr24 (n = 13; A), total FOXO1 (B), FOXO3a (n = 13; C) and LC3b-1 (n = 12; D) at rest following 5 days of EB, 5 days of ED, and a bout of leg press (6 sets × 8 repetitions at 80% 1-RM) in ED with postexercise ingestion of PLA or 30 g of a whey protein drink (PRO).Values are expressed relative to total protein loaded determined by stain-free technology and presented in arbitrary units (means ± SD). Significant difference (P < 0.05) is indicated by (b) vs. ED, (c) vs. PLA 1 h, and (d) vs. PLA 4 h.
ULK1, Atg5, cAtg12, beclin-1, and p62.
There was a main effect of treatment for ULK1Ser757 phosphorylation (Fig. 4A). ULK1 phosphorylation increased in PRO at 1 h postexercise above resting EB and ED (∼30–60%, P < 0.05) and was higher than PRO at 4 h (∼40%, P < 0.05), along with a tendency toward increasing above PLA 4 h (∼35%, P = 0.07) at this time point. There was also tendency for total ULK1 to increase (∼79%, P = 0.089) above ED in PRO at 1 h after REX (Fig. 4B). There were main treatment effects for total levels of Atg5 (Fig. 4C). Atg5 decreased from EB to ED (∼23%, P < 0.001), and these levels remained below EB from 1–4 h postexercise in PLA (∼17%, P < 0.05) and at 1 h in PRO (∼18%, P < 0.05). There were main treatment effects for total levels of cAtg12 protein (Fig. 4D). cAtg12 decreased compared with EB in PLA 4 h (∼20%, P < 0.05), whereas it increased in PRO at 4 h above this time point (∼26%, P < 0.05) to similar values observed in EB. There were no significant differences in Beclin-1 (Fig. 4E) or p62 (Fig. 4F) protein expression. Beclin-1 tended to decrease from EB to ED, but these changes did not reach significance (∼14%, P = 0.083).
Fig. 4.Phospho-ULK1Ser757 (A), total ULK1 (n = 8; B), Atg5 (C), cAtg12 (D), Beclin-1 (E) and p62 (F) at rest following 5 days of EB, 5 days of ED, and a bout of leg press (6 sets × 8 repetitions at 80% 1-RM) in ED with postexercise ingestion of a placebo (PLA) or 30 g of a whey protein drink (PRO).Values are expressed relative to total protein loaded determined by stain-free technology and presented in arbitrary units (mean ± SD). Significant difference (P < 0.05) is indicated by (a) vs. EB, (b) vs. ED, (d) vs. PLA 4 h, and (f) PRO 4 h.
mRNA Expression
Atgs.
There were no significant changes at any time in the mRNA expression of genes with putative roles in autophagy, including Beclin-1, Atg12, Atg4b, GABARAP, LC3b, and the mitophagy marker BNIP3 (Fig. 5).
Fig. 5.Beclin-1 (A), Atg12 (B), Atg4b (C), GABARAP (D), LC3b (E), and BNIP3 (F) at rest following 5 days of EB, 5 days of ED, and a bout of leg press (6 sets × 8 repetitions at 80% 1-RM) in ED with postexercise ingestion of a placebo (PLA) or 30 g of a whey protein drink (PRO). Values are expressed relative to GAPDH and presented in arbitrary units (mean ± SD; n = 12).
SIRT1, FOXO1, PGC-1α, EGR-1, and VEGF.
There were no significant changes in SIRT1, FOXO1, and PGC-1α mRNA expression at any time point (Fig. 6). There was a main effect of treatment for EGR-1 mRNA expression after exercise (Fig. 6D). EGR-1 mRNA increased above EB (PLA, ∼864%; PRO, ∼2,731%, P < 0.001), and ED (PLA, ∼417%; PRO, ∼1,417%, P < 0.001) 1 h post-REX and was higher in PRO at 1 h post-REX than PLA at 4 h (∼136%, P = 0.015). EGR-1 expression in PRO at 4 h was also elevated above EB and ED (∼353–746%, P < 0.01), whereas its expression in PLA at 4 h only increased significantly above EB (∼1,100%, P < 0.05) with a trend toward increasing above ED (∼543%, P = 0.083). Lastly, VEGF mRNA increased significantly above both resting conditions in PLA at 4 h only (∼47–51%, P < 0.05; Fig. 6E).
Fig. 6.SIRT1 (A), FOXO1 (B), PGC-1α (C) EGR-1 (D), and VEGF (E) gene expression at rest following 5 days of EB, 5 days of ED, and a bout of leg press (6 sets × 8 repetitions at 80% 1-RM) in ED with postexercise ingestion of a placebo (PLA) or 30 g of a whey protein drink (PRO). Values are expressed relative to GAPDH and presented in arbitrary units (mean ± SD; n = 12). Significant difference is indicated by (P < 0.05) (a) vs. EB, (b) vs. ED, and (d) PLA 4.
DISCUSSION
The first novel finding of this study was that both short-term ED and REX, when commenced after ED in a fasted state and with no nutrient provision during recovery, failed to increase the transcript abundance, phosphorylation state, and total protein content of several select molecular markers of autophagy or their upstream regulators. In contrast, Atg5 protein levels decreased following short-term ED, whereas consuming protein after REX increased its conjugated form cAtg12 to levels similar in EB, in addition to increasing the total protein abundance of FOXO1 and decreasing p38 MAPK (Fig. 7). Moreover, there were no changes in p53Ser15 phosphorylation or the gene expression of SIRT1 and its deacetylase targets FOXO1 and PGC-1α despite large increases in EGR-1 mRNA abundance postexercise. This study is the first to characterize postabsorptive intramuscular autophagy-regulatory responses following an acute bout of REX undertaken in ED in humans.
Fig. 7.Representative blots for all phosphorylated and total proteins (A) and a Stain-free image of total protein loading (B).
The constitutive turnover of long-lived proteins, polyubiquitinated aggregates and intracellular organelles by autophagy is critical for preserving the integrity of skeletal muscle mass (33). Considering REX modulates MPB (45) and short-term ED (<21 days) can exacerbate MPB responses (9, 10), we investigated the expression of autophagy-regulatory genes and proteins following a single bout of REX undertaken in ED. We observed reduced total protein levels of Atg5 and a trend for attenuated Beclin-1, both proximal effectors of autophagosome biogenesis (55), following 5 days of ED. However, the gene expression, phosphorylation state, and total protein levels of several other molecular markers implicated in autophagy were largely unaffected by ED. We originally hypothesized ED would increase these targets as a means of partitioning the requisite energy substrate for the maintenance of intracellular homeostasis. Although a similar (∼500-1,000 kcal/day) ED in animals (53) and humans (52) has previously been shown to increase the expression of Atgs, the duration of energy restriction in these studies was substantially longer (∼6 mo) than the present investigation. Furthermore, there is evidence to demonstrate that whole-body proteolysis and net protein turnover are attenuated following similar, prolonged restrictions of energy availability (8, 49), which could suggest that a “threshold” duration of ED may be required before autophagy can create a new steady-state net protein balance.
We (2) and others (44) have previously reported 5–10 days of ED to reduce postabsorptive rates of MPS. Notably, this attenuated response was of a similar magnitude to the ED-induced decline in Atg abundance in the present study and may be a consequence of a reduced need for intracellular amino acids otherwise partitioned by autophagy to maintain MPS. Indeed, autophagy-deficient cells have lower protein synthesis due to limited amino acid availability (41). Moreover, eIF2αSer51 phosphorylation, indicative of general protein translational repression (19) and activation of an autophagic transcriptional program under certain stresses (including nutrient deprivation) (3), was unchanged following ED. The present findings could suggest that the acute proteolytic responses to moderate ED are not mitigated by autophagy per se but by the UPS. In this regard, a similar (10 day) ED intervention in healthy adults resulting in a ∼60% increase in MPB was accompanied by elevated caspase-3 protein expression (10), while 21 days of ED increased muscle-specific ubiquitin ligase gene expression (9). Taken collectively, these findings suggest that autophagy may not contribute substantially to muscle proteolysis early in ED. Another consideration is that autophagy, like MPS, is an energy-consuming process (46, 47) and, thus, may have been reduced as a result of reprioritizing ATP for alternate cellular processes. Whether ED-induced changes in MPS regulate or are directly regulated by autophagy is an area for future investigation.
Compared to the dynamic changes reported for the UPS (32, 54), there is a paucity of information addressing how REX modulates autophagy and the time course of this response. Two studies have shown decreases in LC3b-II, but not LC3b-I, following REX in young and old individuals (14) and REX with protein-carbohydrate coingestion (15). Such changes could indicate a reduced autophagic flux after REX, as lipidation of cytosolic LC3b-I to LC3b-II results in the latter localizing to the inner and outer faces of the expanding autophagosome (50). Moreover, as subjects in these studies were in EB, a more substantial energetic perturbation elicited by exercise-diet interactions may have been required to alter the LC3b-II/LC3b-I ratio. In partial support of this notion, we observed reduced LC3b-I protein expression following ED and REX with placebo ingestion. However, this occurred without any commensurate differences in p62, a bridging protein that delivers substrates to the autophagosome via its interaction with LC3b-II (42). Therefore, although our results do not preclude enhanced formation of autophagosomes with REX in ED, future work (e.g., immunoprecipitation of LC3b with p62) in vivo human skeletal muscle is required to confirm such regulation. We did, however, find an increase in the mRNA of the angiogenic marker VEGF above both resting conditions with PLA, occurring alongside the changes in LC3b-I. Given that VEGF-mediated angiogenesis is hypothesized to couple oxygen and nutrient delivery to support contractile activity (1) and that angiogenic adaptation to exercise is attenuated in mice with a genetic disruption of autophagy (27), these results could imply a coordinated autophagic and angiogenic response to mitigate the metabolic demands and subsequent adaptation to REX in ED.
Consuming protein-containing meals during ED can reduce UPS-mediated proteolysis (9), thereby potentially contributing to the “sparing” of muscle mass loss during conditions of negative EB. Insulin has been reported to exert an inhibitory effect toward intramuscular autophagy in mice (39), while in other cell types, amino acid availability restricts autophagic flux (40). Considering our previously reported insulinotrophic effects of whey (2) combined with the aforementioned potential for protein availability to repress the UPS (9), we hypothesized protein ingestion would similarly restrict the exercise-induced increase in autophagy markers in ED. In contrast, REX with PRO partially restored the ED-induced decline in Atg5 by increasing cAtg12 protein abundance above PLA. Similarly, FOXO1, a transcription-independent (56) and -dependent (36) regulator of autophagy also increased with PRO postexercise compared with ED and PLA. It is not entirely clear how PRO mediated increases in the autophagy signaling proteins, as these differences were independent of changes in mRNA transcripts of genes involved in autophagy (including corresponding Beclin-1, Atg12, LC3b, and FOXO1) and the activity of FOXO3a, a transcriptional regulator of Atgs, such as LC3b (31). Desgeorges et al. (12) recently reported an increase Atg protein content following a starvation stimulus in myotubes that occurred concomitantly with the absence of increased gene expression, which led the authors to postulate that these proteins were translated from a preexisting pool of mRNA. Considering postexercise protein ingestion modulates markers of the UPS (3, 5), it is also possible that PRO repressed the activities of novel ubiquitin ligases that tag Atgs for degradation (25). These paradigms represent an important area of future research investigating energy fluctuations and protein transcriptional and/or translational control in ED.
PRO also increased postexercise ULK1Ser757 phosphorylation, a substrate of mTOR that inhibits autophagy induction by disrupting AMPK accessibility to other ULK1 residues (24). This increase in ULK1 phosphorylation may be reflective of a temporal restriction of autophagy until a later postexercise period, as we have previously showed, no differences in AMPK phosphorylation between trials postexercise (2). Nonetheless, the similar responses in MPS (2) and Atg protein abundance with PRO postexercise suggest a restorative effect toward autophagy regulatory processes when exogenous protein is made available in situations of ED. Furthermore, these changes occurred with parallel reductions in total p38 MAPK content, which has been shown to couple pathologically high oxidative stress to autophagy-mediated muscle atrophy (34). Thus, protein ingestion following REX in ED may stimulate autophagy to ultimately promote a positive protein balance. While previous findings demonstrate that exogenous rather than intramuscular amino acid availability in EB is a stronger determinant of MPS (4), the present work highlights that autophagy may contribute to the greater total protein turnover and muscle remodeling processes associated with protein ingestion following REX in ED.
We observed large mRNA increases for the transcription factor EGR-1 following REX independent of protein availability. EGR-1 transcriptionally upregulates SIRT1 in skeletal muscle cells following mechanical stretch (43), and increases in EGR-1 and SIRT1 mRNA have been reported following high-intensity endurance exercise (13). Although SIRT1 regulates autophagy by direct deacetylation of various Atgs (26), our findings suggest that EGR-1 gene regulation following exercise is influenced predominantly by contractile overload rather than energy status. Although we cannot dismiss changes in the total protein content (28), nuclear activity (17), and/or total muscle activity of SIRT1 (16), our results demonstrate that SIRT1 gene expression, along with that of its substrates PGC-1α, FOXO1, and the activity of its additional target p53, is unaffected in human skeletal muscle by short-term ED and REX undertaken in ED; these effects seemingly contrast the signaling response to REX commenced with low glycogen availability (6).
Perspectives and Significance
Autophagy is emerging as a critical mediator for skeletal muscle remodeling and adaptation responses with exercise. We report the novel finding that protein levels of Atg5 are attenuated following short-term ED. However, its activity, as suggested by cAtg12 expression, can be partially “rescued”, commensurate with changes in several other autophagy-regulatory proteins by REX and protein ingestion. Whether this autophagic response is a cause or a consequence of the short-term ED-induced attenuation of MPS requires further investigation. As dysregulation of autophagy is deleterious for skeletal muscle function (33), future studies determining how autophagy signaling is regulated during chronic resistance training with high-protein availability and the impact it may have on the turnover (i.e., repair/remodeling) of muscle proteins should provide valuable mechanistic information for promoting and maintaining skeletal muscle mass.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
Author contributions: W.J.S., J.L.A., V.G.C., S.M.P., D.R.M., T.S., L.M.B., J.A.H., and D.M.C. conception and design of research; W.J.S., J.L.A., and D.M.C. performed experiments; W.J.S., J.A.H., and D.M.C. analyzed data; W.J.S. and D.M.C. interpreted results of experiments; W.J.S. prepared figures; W.J.S. and D.M.C. drafted manuscript; W.J.S., J.L.A., V.G.C., D.R.M., T.S., J.A.H., and D.M.C. edited and revised manuscript; W.J.S., J.L.A., V.G.C., S.M.P., D.R.M., T.S., J.A.H., and D.M.C. approved final version of manuscript.
ACKNOWLEDGMENTS
This study was funded by an Australian Research Council Linkage Project Grant (LP100100010) and an ACU Collaborative Research Network Grant to J. A. Hawley (2013000443). The authors would like to thank Evelyn Parr, Alisa Nana, Louise Cato, Iona Halliday, Greg Shaw, Felicity Galvez, Graeme Allbon, Ryan Kohler, Tom Hilton, Dr. Peter Velloza, and Dr. Andrew Garnham for their technical assistance during clinical trials and laboratory analysis.
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