The functional importance of skeletal muscle autophagy in response to fasting has been suggested to involve the removal and degradation of proteins to provide amino acids for ATP production and/or de novo protein synthesis ( 21 ). Although the blood amino acid concentration at any given time will be determined by both the appearance and disappearance rate of amino acids into the systemic circulation ( 42 ), skeletal muscle autophagy may contribute to changes in plasma amino acid concentrations during fasting. In accordance, fasting for 3 days has been shown to induce specific responses of individual amino acids in the blood, while maintaining the total plasma amino acid concentration ( 11 ). However, combined analyses of the plasma amino acid profile and skeletal muscle autophagy are lacking, and the impact of training state on the fasting-induced changes in the plasma amino acid profile remains to be determined.

Autophagy has been suggested to be regulated by energy deprivation ( 6 , 22 , 23 , 40 ). In addition, increased LC3II-to-LC3I protein ratio and AMPK Thr172 and ULK1 Ser317 phosphorylation as well as decreased p62 protein content and ULK1 Ser757 phosphorylation were observed in skeletal muscle from athletes in response to ~14 h of fasting ( 40 ). Moreover, fasting has been reported mainly to increase AMPK-mediated ULK1 Ser555 phosphorylation in mice ( 6 ), whereas another study showed decreased phosphorylation of ULK1 (Ser 555 ) and acetyl-CoA carboxylase in mouse muscle in response to fasting ( 13 ). Together, these studies indicate that fasting regulates skeletal muscle autophagy. However, a detailed time course of fasting-induced regulation of autophagy in human skeletal muscle is lacking.

Two major nutrient-sensing kinases, AMP-activated protein kinase (AMPK) and mechanistic target of rapamycin (mTOR), have been suggested to regulate autophagy through coordinated phosphorylation of ULK1. AMPK activates ULK1 by phosphorylating several sites including ULK1 Ser317 and ULK1 Ser555 . Furthermore, AMPK is an established negative regulator of the mTOR signaling cascade ( 6 , 37 , 40 ), and mTOR phosphorylates ULK1 at Ser 757 thereby inhibiting autophagy through ULK1 deactivation ( 6 , 20 , 21 ). Thus, studies in cells and mouse skeletal muscle have shown that AMPK was able to relieve mTOR-mediated autophagy repression when nutrient supply was low, allowing activation of autophagy through ULK1 Ser317 or ULK1 Ser555 phosphorylation ( 6 , 9 , 21 ). In addition, it has been demonstrated in cells that mTOR is not completely inhibited by AMPK if amino acids are available and that the residual mTOR activity may prevent ULK1 activation ensuring that autophagy activation is dampened except during severe starvation ( 20 ).

An important step in the initiation of autophagy is the formation of a phagophore double membrane, which matures into an autophagosome containing the components marked for degradation ( 28 ). Phosphatidylethanolamine lipidation of microtubule-associated protein-1A/1B light chain 3 (LC3)I to LC3II is crucial for autophagosomal maturation. Therefore, LC3II is widely used as a marker of autophagosome content, whereas the LC3II-to-LC3I ratio is used as a measure of autophagosome formation ( 18 , 21 , 29 ). Moreover, sequestosome 1/p62 (p62), which targets specific proteins and organelles to the inner autolysosomal membrane, is degraded along with the autolysosome during autophagy and is used as a marker for autolysosomal degradation ( 21 , 39 ). Although protein levels of LC3I, LC3II, and p62 cannot be used as a measure of autophagy flux ( 21 ), these protein markers often used as indicators of changes in autophagy regulation ( 21 ). In addition, recruitment of the Unc-51-like autophagy-activating kinase-1 (ULK1) complex and the class III phosphatidylinositol 3-kinase complex [including, among others, Bcl-2-interacting coiled-coil protein-1 (Beclin1)] is an essential step in the initiation of autophagy ( 36 ). ULK1 and Beclin1 are both likely to act at several stages in the autophagy process and can be regulated through different pathways ( 21 , 34 , 36 ).

Two-way repeated-measures ANOVA was applied to evaluate the effect of fasting and training state on protein content, phosphorylation level, and plasma amino acid levels. One-way repeated-measures ANOVA was applied to further test for the effect of fasting within each training group. Student-Newman-Keuls post hoc test was used to locate differences when the one- and two-way ANOVAs detected main differences. Statistical time effects are given both relative to 2 h after the meal (postprandial) and 12 h after the meal (overnight fast). Differences were considered significant at P < 0.05, and a tendency is reported in results when 0.05 ≤ P < 0.1 to minimize the risk of type 2 errors. Tendencies are not included in the amino acid results. If not otherwise stated, values are presented as means ± SE. SigmaPlot 13.0 was used as statistical software (Systat Software).

Protein content and phosphorylation of the given proteins were measured in muscle lysates by SDS-PAGE, loading equal amounts of protein on hand-casted gels with appropriate acrylamide percentages (Tris·HCl 6–15%), and Western blot analysis, using PVDF membranes (Immobilon-P Transfer membranes; Millipore) and semidry transfer, as previously described ( 4 ). Membranes were blocked in either 3 or 5% fish gel (Sigma) in Tris-buffered saline-Tween 20, followed by membrane incubation with primary antibody (1:1,000 in 5% BSA). The following primary antibodies were used: AMPKα1 (kindly provided by Prof. Grahame Hardie, University of Dundee, Dundee, Scotland), AMPKα Thr172 (no. 2535-S), AKT1 (no. 2967), AKT Ser473 (no. 9271-S), AKT Thr308 (no. 9275-S), Beclin1 (no. 3738), Beclin1 Ser93 (no. 14717), LC3I + LC3II (no. 4108), mTOR Ser2448 (no. 2971), ULK1 (no. 8054), ULK1 Ser317 (no. 12753), ULK1 Ser757 (no. 6888), and ULK1 Ser555 (no. 5869), all from Cell Signaling Technology, as well as p62 (no. ab-56416) and p21 (no. ab-109199) from Abcam. Membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibody (in 3 or 5% fish gel + Tris-buffered saline-Tween 20) and developed with Luminata Classico or Forte Western HRP substrate (Millipore), and bands were visualized with a luminescent image analyzer (ImageQuant LAS 4000; GE Healthcare Life Sciences, Little Chalfont, United Kingdom). The results were quantified in ImageQuant TL software (GE Healthcare). Protein content of specific proteins was expressed as arbitrary units relative to control samples loaded on each gel, and equal loading and blotting were confirmed by a similar p21 protein content in all groups. In addition, membranes were blue stained after target protein measurements to further confirm equal protein loading. For each specific protein, a series of different standard loads was included to ensure that protein analyses were performed in the linear range. Representative blots of trained and untrained subjects for a given protein are from the same membrane.

In the morning 2 h before the first muscle biopsy, subjects received a standardized meal (caloric content: 42% carbohydrate, 12% protein, and 46% fat). Skeletal muscle biopsies [and abdominal subcutaneous adipose tissue biopsies for another purpose ( 3 )] were obtained from the middle portion of the vastus lateralis muscle, using the percutaneous needle biopsy technique ( 2 ) with suction under local anesthesia at 2, 12, 24, and 36 h after the standardized meal. The skeletal muscle biopsies were quick-frozen in liquid nitrogen and stored at −80°C for later analyses. Additionally, blood samples were obtained from an arm vein at each sampling time point. Blood was collected in EDTA-containing tubes, and plasma was obtained by centrifugation. Plasma samples were stored at −80°C for later analyses. The subjects were asked to refrain from strenuous activity during the fasting period as well as minimize daily physical activity during the fasting period.

V̇o 2max was determined on a cycle ergometer using an incremental bicycle test. Subjects who self-reported as untrained performed a protocol where they rested on the cycle ergometer for 1 min followed by 5 min at 85 W of cycling, 5 min at 140 W, and a graded load increase of 20 W every minute until exhaustion. Likewise, subjects who self-reported as trained performed a protocol where they rested on the cycle ergometer for 1 min followed by 5 min at 125 W of cycling, 5 min at 200 W, and a graded load increase of 25 W every minute until exhaustion.

Initially, 17 healthy male subjects were recruited on the basis of determination of whole body maximal oxygen uptake (V̇o 2max ). Inclusion of subjects required either a V̇o 2max below 45 ml·min −1 ·kg −1 (untrained) or a V̇o 2max above 55 ml·min −1 ·kg −1 (trained). Further inclusion criteria was defined based on skeletal muscle oxidative capacity. Hence, citrate synthase activity and oxidative phosphorylation complex protein content were measured in skeletal muscle biopsies obtained at rest as markers of oxidative capacity to establish two nonoverlapping populations of untrained and trained individuals ( 14 ). This led to the exclusion of four subjects and resulted in seven untrained and six trained individuals, with no significant differences between the two groups in the following parameters: age 28 ± 3 and 27 ± 4 yr, height 183 ± 10 and 183 ± 4 cm, body weight 89 ± 19 and 79 ± 5 kg, and body mass index 26 ± 1 and 24 ± 1 (means ± SD) for untrained and trained subjects, respectively. The study was approved by the Ethics Committee of Copenhagen and Frederiksberg Communities (H-15010768) and was conducted in accordance with the guidelines of the Declaration of Helsinki. All participants provided written informed consent. Tissue and blood samples from the present experiment have been used to address other research questions ( 3 , 14 ). The inclusion criteria used by Bertholdt et al. ( 3 ) were based solely on V̇o 2max performance because of use of adipose tissue in contrast to skeletal muscle in the present study.

Marked changes were seen in the concentrations of several nonessential amino acids. The plasma proline concentration was ~30% lower ( P < 0.05) at 12, 24, and 36 h than at 2 h after the meal in both untrained and trained subjects and ~20% lower ( P < 0.05) at 24 and 36 h than at 12 h after the meal in untrained subjects. The plasma proline concentration was ~30% lower ( P < 0.05) in trained than untrained subjects at 2, 12, 24, and 36 h after the meal ( Table 2 ). The plasma aspartic acid concentration was ~20% lower ( P < 0.05) at 36 h than at 2 and 12 h after the meal in untrained subjects and was ~25% lower ( P < 0.05) at 12 and 36 h than at 2 h after the meal in trained subjects. Moreover, plasma aspartic acid concentration was ~35% lower ( P < 0.05) in trained than untrained subjects at 2, 12, and 24 h after the meal ( Table 2 ). There was no effect of fasting on plasma glutamic acid concentration in untrained subjects, whereas plasma glutamic acid concentration was ~45% lower ( P < 0.05) at 24 and 36 h than at 2 h after the meal in trained subjects. Plasma glutamic acid concentration was ~50% lower ( P < 0.05) in trained than untrained subjects at 24 h after the meal ( Table 2 ). The plasma tyrosine concentration was ~15% lower ( P < 0.05) at 24 h than at 2 and 12 h after the meal in untrained subjects, whereas there was no effect of fasting on plasma tyrosine in trained subjects. Moreover, the plasma tyrosine concentration was ~20% lower ( P < 0.05) in trained than untrained subjects at 2 and 12 h after the meal ( Table 2 ). The plasma tau-methylhistidine concentration was ~30% lower ( P < 0.05) at 24 h than at 2 and 12 h after the meal. There was no effect of training state on plasma tau-methylhistidine ( Table 2 ). There was no effect of fasting in either the untrained or the trained subjects or of training state on the plasma alanine, asparagine, glutamine, and serine concentrations ( Table 2 ).

Overall, there was no effect of fasting or training state on the plasma concentrations of the essential amino acids (histidine, lysine, methionine, phenylalanine, or threonine) either in the untrained or the trained subjects, the only exception being plasma tryptophan, where the plasma concentration was ~20% lower ( P < 0.05) in trained than untrained subjects at 36 h after the meal ( Table 2 ). Plasma leucine, isoleucine, and valine, the branched-chain amino acids (BCAA), behaved similarly ( Fig. 5 B and Table 2 ) with ~35% higher ( P < 0.05) concentrations at 36 h than at 2 and 12 h after the meal in both untrained and trained subjects. However, the BCAA concentrations were ~15% lower ( P < 0.05) in trained than untrained subjects at 24 h after the meal.

There was an overall tendency for Beclin1 Ser93 phosphorylation/Beclin1 protein to change ( P = 0.095) with fasting time, whereas there was no difference in skeletal muscle Beclin1 Ser93 phosphorylation between time points either in untrained or trained subjects. Skeletal muscle Beclin1 Ser93 phosphorylation/Beclin1 protein was ~70% lower ( P < 0.05) in trained than untrained subjects, at 24 h after the meal, whereas there was no difference in Beclin1 Ser93 phosphorylation between untrained and trained subjects ( Table 1 and Fig. 3 ).

There was a significant interaction ( P < 0.05) between fasting time and training state in skeletal muscle Beclin1 protein content. Beclin1 protein content was ~1.3-fold higher ( P < 0.05) at 12 h than at 2 h after the meal and lower ( P < 0.05) at 36 h than at 12 h after the meal in trained subjects, as well as ~40% lower ( P < 0.05) at 24 h than at 2 and 12 h after the meal in untrained subjects. Moreover, Beclin1 protein content was ~1.7-fold higher ( P < 0.05) in trained than untrained subjects at 12 and 24 h after the meal ( Figs. 2 E and 3 ).

Fig. 2. Unc-51-like autophagy-activating kinase-1 (ULK1) Ser757 phosphorylation ( A ), ULK1 Ser757 phosphorylation normalized to ULK1 protein content (ULK1 Ser757 phos/ULK1 protein, B ), ULK1 Ser555 phosphorylation ( C ), ULK1 Ser555 phos/ULK1 protein ( D ), and Bcl-2-interacting coiled-coil protein-1 (Beclin1) protein ( E ) in vastus lateralis muscle from untrained and trained subjects 2, 12, 24, and 36 h after a standardized meal. Representative blots of all the proteins and phosphorylations are shown for one untrained and one trained subject in Fig. 3 . Protein and phosphorylation levels are given in arbitrary units (AU). Values are means ± SE; n = 6–7, except ULK1 Ser555 phos/ULK1 protein ( n = 5–6). *Significantly different from 2-h fasting within given training group, P < 0.05. ¤Significantly different from 12-h fasting within given training group, P < 0.05. #Significantly different from untrained group within given fasting time point, P < 0.05. There is a significant interaction between training state and time of fasting in ULK1 Ser757 phosphorylation and Beclin1 protein content, P < 0.05.

There was a significant interaction ( P < 0.05) between fasting time and training state in skeletal muscle ULK1 Ser757 phosphorylation. In trained subjects, ULK1 Ser757 phosphorylation was ~55% lower ( P < 0.05) at 36 h than at 2 h after the meal, whereas there was no difference in ULK1 Ser757 phosphorylation between time points in untrained subjects. ULK1 Ser757 phosphorylation tended to be higher ( P = 0.054) and was ~2.3-fold higher ( P < 0.05) in trained than untrained subjects at 2 and 24 h after the meal, respectively ( Figs. 2 A and 3 ). There was an overall tendency for ULK1 Ser757 phosphorylation/ULK1 protein to decrease ( P = 0.077) with fasting duration, and there was no difference between untrained and trained subjects ( Figs. 2 B and 3 ).

There was a significant interaction ( P < 0.05) between fasting time and training state in skeletal muscle AMPK Thr172 phosphorylation and AMPK Thr172 phosphorylation/AMPKα1 protein. Moreover, AMPK Thr172 phosphorylation and AMPK Thr172 phosphorylation/AMPKα1 protein in trained subjects were ~1.7-fold higher ( P < 0.05) at 12 and 24 h than at 2 h after the meal and ~70% lower ( P < 0.05) at 36 h than at 2 h. In addition, AMPK Thr172 phosphorylation was ~70% lower ( P < 0.05) at 36 h than at 12 h after the meal in trained subjects. There was no difference between any of the time points within the untrained subjects. AMPK Thr172 phosphorylation tended to be higher ( P = 0.073) in trained than untrained subjects at 2 h after the meal, and AMPK Thr172 phosphorylation as well as AMPK Thr172 phosphorylation/AMPKα1 protein were ~3- and ~5-fold higher ( P < 0.05) at 12 and 24 h, respectively, in trained than untrained subjects ( Figs. 1, E and F , and 3 ).

Fig. 1. AKT Thr308 phosphorylation ( A ), AKT Thr308 phosphorylation normalized to AKT1 protein content (AKT Thr308 phos/AKT1 protein, B ), mechanistic target of rapamycin (mTOR) Ser2448 phosphorylation ( C ), mTOR Ser2448 phos/mTOR protein ( D ), AMP-activated protein kinase (AMPK) Thr172 phosphorylation ( E ), and AMPK Thr172 phos/AMPKα1 protein ( F ) protein in vastus lateralis muscle from untrained and trained subjects 2, 12, 24, and 36 h after a standardized meal. Representative blots of all the proteins and phosphorylations are shown for one untrained and one trained subject in Fig. 3 . Phosphorylation levels are given in arbitrary units (AU). Values are means ± SE; n = 4–6 ( A and D ), n = 5–6 ( B ), n = 6–7 ( C and E ), and n = 5–7 ( F ). *Significantly different from 2-h fasting within given training group, P < 0.05. ¤Significantly different from 12-h fasting within given training group, P < 0.05. #Significantly different from untrained group within given fasting time point, P < 0.05. There is a significant interaction between training state and time of fasting in AMPK Thr172 phos and AMPK Thr172 phos/AMPKα1 protein, P < 0.05.

AKT Thr308 phosphorylation and AKT Thr308 phosphorylation normalized to AKT1 protein content (AKT Thr308 phosphorylation/AKT1 protein) in skeletal muscle were ~50–80% lower ( P < 0.05) at 12, 24, and 36 h than at 2 h after the meal in untrained subjects. In trained subjects, AKT Thr308 phosphorylation tended to be lower ( P = 0.059) at 36 h than at 2 h after the meal, whereas there was no difference in AKT Thr308 phosphorylation/AKT1 protein between fasting time points in trained subjects. AKT Thr308 phosphorylation was ~2.5–3-fold higher ( P < 0.05) in trained than untrained subjects at 12 and 24 h after the meal, whereas there was no difference in AKT Thr308 phosphorylation/AKT1 protein between trained and untrained subjects ( Figs. 1, A and B , and 3 ).

DISCUSSION

The main findings of the present study are that 36 h of fasting reduced LC3I, LC3II, and p62 protein content only in skeletal muscle from untrained subjects. However, AMPKThr172 phosphorylation, ULK1Ser555 phosphorylation, and Beclin1 protein content were higher, whereas ULK1Ser757 phosphorylation was lower, in skeletal muscle from trained than untrained subjects during fasting indicating that training state influences autophagy signaling during fasting.

The present observation that fasting decreased LC3II protein content in skeletal muscle from the untrained subjects is opposite of the reported marked increase in skeletal muscle LC3II protein content with fasting in humans with unknown training state (43) and mice (17, 27, 29). The different LC3II protein response between the previous studies and the present human study may be due to muscle type (24, 29) or be related to the duration of fasting, because humans in the previous study fasted for 72 h and the decrease in LC3II protein content in the present study was observed at 12 and 24 h of fasting only (1, 43). Moreover, it is possible that a transient increase in LC3II protein content preceded the decrease observed in the untrained subjects in the present study as observed in mouse embryo fibroblast (MEF) cell culture after 0.5 h (30) or later as previously observed at 72 h of fasting in humans (43). On the other hand, a robust increase in autophagy flux was reported in mouse skeletal muscle at both 24 and 48 h of fasting (27, 29), suggesting that the observed dissimilarities may be due to different regulation in mice and humans, although 48 h of fasting for mice is very extreme considering the high metabolic rate of mice (35). Increases in LC3II protein content are typically interpreted as an increased number of autophagosomes (29, 30), and the observed decline in LC3II protein content may therefore indicate that fasting reduced the number of autophagosomes in skeletal muscle of the untrained subjects. This possibility is supported by the transient decrease of Beclin1 protein content together with unchanged absolute and normalized levels of ULK1Ser555, ULK1Ser317, and AMPKThr172 phosphorylation in skeletal muscle from untrained subjects in response to fasting. On the other hand, the finding that p62 protein content decreased in the untrained subjects with fasting may be interpreted as increased removal of p62 by autophagy. However, whether these changes in autophagy-related protein contents reflect removal by autophagy or decreased synthesis remains to be determined, for example, by using autophagy inhibitors.

The unchanged absolute and normalized levels of AMPKThr172 phosphorylation in untrained subjects in response to fasting are in line with observations in humans at 8, 16, 24, or 48 h of fasting (44, 45), whereas an increase in AMPKThr172 phosphorylation has been reported in untrained humans after 72 h of fasting (1). The finding that absolute and normalized levels of mTORSer2448 phosphorylation did not change supports an unchanged mTOR-mediated autophagy in untrained subjects, confirming one study (44) but contradicting two other human studies that observed a decrease in mTOR phosphorylation (1, 43). An explanation for these differences in AMPKThr172 and mTORSer2448 phosphorylation may be the duration of fasting because subjects fasted for 72 h in the previous studies (1, 43) and only 36 h in the present study. Alternatively, these differences may be ascribed to whether the response is relative to an overnight fast or to the postprandial state. The present observation that LC3I protein content also decreased during fasting in the untrained subjects either indicates that more LC3I was converted to LC3II or that less LC3I was produced. The indications that autophagy signaling did not change in the untrained subjects in response to fasting may suggest that the decline in LC3I, LC3II, and p62 protein contents was due to reduced synthesis rather than enhanced removal by autophagy, although it cannot be excluded that circadian rhythm influences the responses to fasting as previously suggested in mice (26).

The present observation that total plasma amino acid levels did not change with 36 h of fasting is corroborated by a study reporting that total plasma amino acid levels were unaffected by 40 days of fasting in obese human subjects (11). Furthermore, the increases in the concentrations of the BCAA leucine, isoleucine, and valine, as well as decreases in aspartic acid and proline concentrations, at 36 h are in accordance with a previous study (11), whereas the decline in plasma alanine concentration at 36 h of fasting was not as convincing as in previous studies (10, 11, 33). These findings indicate either increased protein breakdown and/or decreased protein synthesis in skeletal muscle during fasting, which needs to be investigated with further studies using isotopic tracers. However, the observations that changes in plasma amino acid concentrations occurred without clear indications of increased autophagy in skeletal muscle suggest that skeletal muscle autophagy did not contribute to the changes in plasma amino acid concentrations in the present study.

The present findings that p62, LC3I, and Beclin1 protein content was similar in skeletal muscle from trained and untrained subjects 2 h after the standardized meal are not in accordance with the previous observations in mouse skeletal muscle in response to endurance exercise training (24). On the other hand, the observation that fasting had no effect on LC3I, LC3II, p62, or Beclin1 protein content in skeletal muscle from trained subjects despite the decline in untrained subjects indicates a training state-dependent regulation of LC3, p62, and Beclin1 protein. Similarly, the change in ULK1 and Beclin1 protein content as well as AMPKThr172, ULK1Ser757, and ULK1Ser555 phosphorylation during fasting only in trained subjects and the observed different levels of these autophagy-related factors in trained and untrained subjects during fasting further suggest that training state influences markers of autophagy in human skeletal muscle in response to fasting. However, it is noteworthy that the observed higher levels of ULK phosphorylation may be driven by higher levels of ULK1 protein content in the trained subjects. The observed higher Beclin1 protein content in trained than untrained subjects during fasting is in line with previous observations that Beclin1 protein content increased with exercise training in rodents (12, 24). Likewise, the present findings that ULK1 protein content and AMPKThr172 phosphorylation were transiently increased with fasting in skeletal muscle of the trained subjects are in accordance with the observation that ULK1 protein content increased in humans after 72 h of fasting (1) and that AMPKThr172 and ULK1Ser555 phosphorylation increased in mouse muscle with 48 h of fasting (6). Furthermore, a study performed in experienced cyclists and triathletes showed no change in muscle AMPKThr172 phosphorylation at 9.5–14 h of fasting compared with 8 h of fasting (40). As AMPK activation has been reported to promote autophagy in skeletal muscle (6, 9, 20), the observed increase in AMPKThr172 phosphorylation in the trained subjects would be expected to be associated with enhanced autophagy in the trained subjects at 12 and 24 h of fasting, and the higher phosphorylation of ULK1Ser555 in trained than untrained subjects supports this. Moreover, Beclin1 is a known ULK1 target (34), and ULK1 kinase activity has been suggested to be necessary for Beclin-mediated autophagy signaling during amino acid withdrawal in MEFs (34, 36). The lack of change in LC3II protein content, LC3II-to-LC3I ratio, and p62 protein content as well as Beclin1Ser93, ULK1Ser317, and ULK1Ser555 phosphorylation with fasting in the trained subjects is supported by the previous suggestion that AMPK-mediated Beclin1Ser93 phosphorylation is required for autophagy induction during glucose starvation in MEFs (19). Thus, AMPK activation may be involved in inducing autophagy (9, 20, 38) but does not seem to be sufficient to elicit changes in autophagy markers in trained subjects in the present study. The training state difference between the present study and the previous study (40) may also to some degree be explained by either the type of exercise performed or the fact that the trained and untrained subjects in the present study were included on the basis of skeletal muscle oxidative capacity in addition to whole body V̇o 2max , whereas the other study used V̇o 2max (40).

The present observation that absolute and normalized levels of AKTThr308 phosphorylation were reduced in skeletal muscle with fasting in untrained subjects is in line with the decline in plasma insulin levels (3). Moreover, the lack of change in AKTThr308 phosphorylation in trained subjects with fasting and the higher AKTThr308 phosphorylation level despite lower plasma insulin level in the trained than the untrained subjects, as previously reported (3), support a higher insulin sensitivity in trained than untrained subjects at least until 24 h of fasting, as anticipated (8, 41). However, these differences in AKTThr308 phosphorylation were not clearly reflected in the downstream AKT target mTOR, indicating that other factors also regulate mTORSer2448 phosphorylation. Furthermore, the finding that the BCAA as well as proline, aspartic acid, glutamic acid, tryptophan, and tyrosine concentrations in the present study were lower in trained than untrained subjects has not been shown previously. In addition, the present observations that the plasma profile of several amino acids was different in untrained and trained subjects have not been shown before and indicate that plasma amino acid appearance and/or disappearance in response to fasting is affected by training state. Future studies using stable isotope-labeled amino acids are needed to clarify in vivo human systemic and skeletal muscle protein synthesis and degradation in untrained and trained subjects in response to fasting.

In conclusion, phosphorylation and protein levels of several autophagy-related proteins were higher in trained than untrained subjects, and fasting reduced LC3I, LC3II, and p62 protein content in skeletal muscle of untrained subjects but not trained subjects. Thus, fasting did elicit a response in several markers of autophagy, but without indications of initiation of autophagy in untrained human skeletal muscle. In addition, several plasma amino acids were different in untrained and trained subjects during fasting. Taken together, the findings of the present study suggest that 36-h fasting regulates autophagy in human skeletal muscle as well as plasma amino acids in a training state-dependent manner.