Regulatory Coordination between Two Major Intracellular Homeostatic Systems

Cells

A549 cells (human lung epithelial carcinoma cell line), Caco-2 cells (colorectal adenocarcinoma), and NCI-H1299 cells (human non-small cell lung cancer; ATCC catalog no. CRL-5803^TM) were purchased from the American Type Culture Collection (Manassas, VA) and maintained at 37 °C in a culture medium composed of DMEM with 4.5 mg/ml glucose, 50 units/ml penicillin, 50 units/ml streptomycin, 4 mm glutamine, and 25 mm HEPES and supplemented with heat-inactivated 10% fetal calf serum that was purchased from Invitrogen. Stock cultures were maintained in 100-mm dishes and were subcultured every 3–4 days. Cultures for all assays were maintained in 35-mm tissue culture plates (Corning Inc., Corning, NY).

Chemicals

Rapamycin was purchased from LC Laboratories (Woburn, MA). LC3 and β-actin antibodies were purchased from Sigma. HSP70 and HSF1 antibodies were obtained from StressGen Biotechnologies (Victoria, British Columbia, Canada). CHOP and eIF2α (total and phosphor) antibodies were purchased from Cell Signaling Technology (Danvers, MA). XBP-1 antibody was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX). Triton X-100, bovine serum albumin, normal donkey serum, and anti-β-actin antibody were purchased from Sigma. Horseradish peroxidase (HRP)-conjugated secondary antibodies for Western blot analysis were purchased from Cell Signaling Technology (Danvers, MA). Cy-5 antibodies for immunostaining were purchased from Jackson ImmunoResearch (West Grove, PA). Tween 20 and nonfat dry milk were purchased from Bio-Rad. All other chemicals were of reagent grade and were purchased from Sigma, VWR (West Chester, PA), or Fisher.

HSP70 Overexpression

As described previously (30), the HSP70 coding region was amplified from pRc/RSV-HSP72 provided by Dr. Anne Knowlton (University of California, Davis) using primer sets that were designed to add HindIII and XbaI sites to the 5′ and 3′ ends, respectively, and inserted into the pRc/CMV vector to construct a vector termed RP70. In a similar manner, the coding region was amplified using primers to add NotI and XbaI sites and cloned into pAdTrack (Ad70). Recombination with the adenovirus backbone was performed in BJ5183-AD-1 cells (Stratagene, La Jolla, CA), and the resulting construct was transfected into 293 cells to produce virus directing the expression of exogenous HSP70. Recombination with the parent pAdTrack (AdTrack) plasmid created the construct used as a control in all experiments. Cells were infected either with control adenovirus or adenovirus directing the expression of HSP70 (a total dose of 2.5 × 10¹⁰ IFUs per plate) for 72 h.

Site-directed Mutagenesis of HSP70

Site-directed mutagenesis of HSP70 was performed according to the to the manufacturer's instruction manual (Agilent Technologies, Inc. Santa Clara, CA).

siRNA of HSF-1

As described previously (29), to silence HSF-1, ON-TARGETplus SMARTpool (Dharmacon, Inc., Chicago) was used. The sequences for HSF-1 small interfering RNA (siRNA) were as follows: 5′-PUACUUGGGCAUGGAAUGUGUU-3′; 5′-PGUCCAUAGCAUCCAAGUGGUU-3′; 5′-PUAUGUCUUCACUCUUCAGGUU-3′; and 5′-PUGAAUCCGGGCUGCUGUUCUU-3′. A549 cells monolayers were transiently transfected using DharmaFect transfection reagent (Dharmacon, Lafayette, CO). A549 cells were seeded into a six-well plate and grown to 80% confluency. A549 monolayers were then washed with PBS, and Opti-MEM medium was added to the well. The plasmid vector containing the siRNA of HSF-1 and DharmaFect reagent was preincubated in Opti-MEM. After 5 min of incubation, two solutions were mixed and incubated for another 20 min, and the mixture was added to each well. After incubation for 3 h at 37 °C, 500 μl of DMEM containing 10% FBS and no antibiotics were added to cell culture media to reach a 2.5% final concentration of FBS. The siRNA-induced silencing of HSF-1 was confirmed by immunoblot of HSF-1.

Quantitative RT-PCR

The total RNA was isolated from A549 cells using RNeasy kit (Qiagen), and cDNA was generated using QuantiTect reverse transcription kit (Qiagen) as per the manufacturer's instructions. The quantitative RT-PCR was performed using SYBR Green I QuantiFast SYBR Green kit (Qiagen) as per the manufacturer's instructions using the following amplification conditions: PCR initial activation step, 95 °C for 5 min; two-step cycling, 40 cycles of denaturation, 95 °C for 10 s; combined annealing/extension, 60 °C for 10 s. The primers for p62 were as follows: forward primer, 5′-cacctgtctgagggcttctc-3′; reverse primer, 5′-agtttcctggtggacccatt-3′. The results were analyzed using relative quantification by comparing ratios of p62 gene and reference housekeeping gene, β-actin (internal control gene). Relative quantification of expression of the p62 gene was studied, and the fold change in expression of p62 gene was calculated relative to β-actin. The mean fold change in expression of the p62 gene was calculated using 2^−ΔΔCT method.

Western Blot Analysis

For LC3 and HSF-1 activation determined by Western blot analysis and binding assay, cells were harvested and nuclear extracts were prepared. To study the effect of HSP70 protein overexpression on macroautophagy, A549, Caco-2, or NCI-H1299 monolayers were infected with the adenovirus (a total dose of 2.5 × 10¹⁰ IFUs per plate) for 3 days followed by starvation in Earle's balanced salt solution (EBSS) for 2 h. Western blot analysis was performed as described previously (28); at the end of the experimental period, monolayers were immediately rinsed with ice-cold PBS, and cells were lysed with lysis buffer (50 mm Tris·HCl, pH 7.5, 150 mm NaCl, 500 μm NaF, 2 mm EDTA, 100 μm vanadate, 100 μm PMSF, 1 μg/ml leupeptin, 1 μg/ml pepstatin A, 40 mm para-nitrophenyl phosphate, 1 μg/ml aprotinin, and 1% Triton X-100) and scraped, and the cell lysates were placed in microcentrifuge tubes. Cell lysates were centrifuged to yield a clear lysate. Supernatant was collected, and protein measurement was performed using the protein assay kit from Bio-Rad. Laemmli gel loading buffer was added to the lysate containing 5–10 μg of protein and boiled for 7 min, after which proteins were separated on an SDS-polyacrylamide gel. Proteins from the gel were transferred to the membrane (Trans-Blot Transfer Medium, Nitrocellulose Membrane; Bio-Rad) overnight. The membrane was incubated for 2 h in blocking solution (5% dry milk in TBS/Tween 20 buffer). The membrane was incubated with appropriate primary antibodies in blocking solution. After being washed in TBS/Tween buffer, the membrane was incubated in appropriate secondary antibodies and developed using the Santa Cruz Western blotting luminol reagents (Santa Cruz Biotechnology, Santa Cruz, CA) on the Kodak BioMax MS film (Fisher).

Quantitative p62 Puncta Analysis

Endogenous p62 puncta were detected by indirect immunofluorescence using a mouse anti-p62 antibody (BD Biosciences) that was detected by an AlexaFluor568-conjugated anti-mouse secondary antibody (Invitrogen). Stained samples were imaged using Cellomics Array Scan (Thermo Scientific, Rockford, IL) in which 49 fields were collected per well in 96-well plates. The mean number of p62 puncta per infected (GFP-positive) cell was then determined by a puncta-counting application integrated within the iDev software (Thermo Scientific).

Immunostaining of LC3 and HSP70 Proteins

For immunostaining of LC3 and HSP70 proteins, monolayers grown on coverslips were exposed to appropriate experimental conditions. As described previously (29), at the end of the experimental period, cells were washed twice in cold PBS and were fixed with 2% paraformaldehyde for 20 min. Then cells were permeabilized with 0.1% Triton X-100 in PBS at room temperature for 20 min. The monolayers were then incubated in blocking solution composed of bovine serum albumin and normal donkey serum in PBS for 1 h. Cells were then labeled with primary antibodies in blocking solution overnight at 4 °C. After being washed with PBS, the coverslips were incubated in appropriate Cy-5-conjugated secondary antibody for 1 h at room temperature and mounted on microscope slides (Erie Scientific, Portsmouth, NH). Immunolocalizations of TJ proteins were visualized using a Nikon fluorescence microscope (Nikon, Garden City, NY) equipped with a Hamamatsu digital camera (Hamamatsu Photonics, Hamamatsu, Japan). Images were processed with Wasabi software (Hamamatsu Photonics Deutschland, Herrsching, Germany).

Induction of Thermal Stress

To induce thermal stress, cells were grown on 35-mm plates and placed in a bath in a manner that the dishes rested directly on water that was heated to 42 °C. To maintain 5% CO₂ concentration during heat exposure, air containing CO₂ was circulated through the sealed bath.

Exercise Protocol and Glutamine Supplementation

Eight total endurance-trained adult men and women (age 18–45) who suffer from exercise-induced gastrointestinal distress were recruited from the University population and local running clubs. All subjects completed a health questionnaire, and a full explanation of procedures, discomforts, and risks were explained before written informed consent was obtained. Selection criteria included a report of gastrointestinal symptoms on at least five occasions during exercise and currently participating in an endurance run training program. Potential subjects were excluded if taking medications (antidepressants) or nutritional supplements. All testing was performed in the exercise laboratory at the University of New Mexico at 1524 m of altitude.

Each subject participated in a double blinded glutamine (Gln) and placebo trial, separated by a 4-week washout period. Subjects ingested 0.3 g/kg of fat-free mass, three times per day for 7 days of Gln or sugar-free lemon flavored placebo. The powder was separated into three doses that were taken in the morning, early afternoon, and evening. Supplements were mixed with water prior to drinking. Prior to the first exercise trial, subjects were provided a 7-day sample bag containing Gln or placebo. Instructions were provided detailing the proper way to take the supplement. At the end of the 7 days of supplementation, subjects performed the exercise trial.

Base-line measurements (maximal oxygen consumption, body composition, blood analysis, and gut permeability) were performed on each subject prior to the start of the study. On the day of the first exercise trial, subjects reported to the lab after an overnight fast. Height and weight were measured along with insertion of a rectal probe for core temperature measurement. The exercise was a 60-min treadmill run at 70–80% of VO_{2 max} in a warm environmental chamber (30 °C) and with a termination criterion of 40 °C. Venous blood was collected at four time points during each exercise trial, including rest, immediately post exercise, 2 h post exercise, and 4 h post exercise.

For base-line measurements, peak aerobic power capacity (VO_{2 peak}) was measured using open circuit spirometry (Parvomedics, Sandy, UT) and a graded incremental protocol on a motorized treadmill. The highest value for oxygen consumption based on an 11-breath running average was used to assess VO_{2 peak}. Additional criteria, including plateau (<150 ml), respiratory exchange ratio (>1.15), rating of perceived exertion (>17), and heart rate (±10 beats/min of max), were assessed to determine whether a true maximal effort was given. Skinfold calipers were used to measured body density by calculating the sum of three sites (men: chest, abdomen, thigh; women: triceps, suprailliac, thigh), and fat-free mass was calculated using the Siri equation. After 20 min of seated rest, a 20-ml blood sample was taken to measure base-line plasma levels of glutamine, along with PBMC levels of LC3 and HSP70.

Statistical Analysis

Results are expressed as means ± S.E. Statistical significance of differences between mean values was assessed with Student's t tests for unpaired data. All reported significance levels represent two-tailed p values. A p value of <0.05 was used to indicate statistical significance.

Effect of HSP70 Protein Expression on Starvation-induced Autophagy

Microtubule-associated protein light chain 3 (LC3) immunoblotting was widely used to monitor the autophagy. Autophagy is manifested by conversion of the unconjugated cytosolic form of LC3-I to the phosphatidylethanolamine-conjugated form of LC3-II that targets to the autophagosomal membrane (37, 38). In the following studies, A549, H1299, or Caco-2 cells were infected with either control adenovirus or adenovirus directing expression of HSP70 (2.5 × 10¹⁰ IFUs per plate). Three days later, starvation was performed by adding EBSS to the cells along with bafilomycin A1 (Baf) (100 nm) for 1 or 2 h (39). Full medium with Baf (100 nm) was added to cells under control conditions. Cells were harvested and lysed, and aliquots were used to determine HSP70 and LC-3. The LC-3 densitometric values were normalized to β-actin protein expression as a commonly used procedure (40, 41). There was a marked increase in HSP70 protein expression in the cells infected with Ad70 compared with the cells infected with a control adenovirus (Fig. 1). At 1 h of starvation, in A549 cells there was a small and insignificant increase in LC3-II protein expression (Fig. 1, A and D). Two hours after starvation, there was a significant increase in LC3-II levels indicating an increase in autophagy. The magnitude of change is consistent with the routinely reported experimental values in the autophagy literature and summarized by Mizushima et al. (41) and Klionsky et al. (40). Interestingly, HSP70 overexpression prevented this starvation-induced autophagy, suggesting that HSP70 plays a regulatory role in modulation of autophagy. Similar effect of HSP70 on autophagy was seen in H1299 and Caco-2 cells (Fig. 1, B, C, E, and F). During autophagy induction, phosphatidylethanolamine-conjugated form of LC3 targets autophagosomal membrane resulting in the formation of punctate organelles that can be quantified by fluorescence microscopy (37). To assess the effect of HSP70 on LC3 puncta formation, A549 cells grown on coverslips were transfected with RFP-LC3. After 1 h, GFP-adenovirus (either control or overexpressing HSP70) was added to the experimental media. Following a 3-day incubation, cells were exposed to starvation conditions (EBSS with 100 nm Baf for 2 h) to induce autophagy. Cells were immunostained for puncta formation. Upon starvation, cells expressing GFP-LC3 showed a transition from diffusive cytoplasm pattern to the punctated membrane pattern, indicating the formation of autophagic vacuoles (Fig. 1, G and H). Treatment of cells with adenovirus overexpressing HSP70 decreased the number of puncta, suggesting a suppressed formation of LC3 and the translocation to the autophagosomes. In the following experiments, the effect of HSP70 overexpression on rapamycin-induced autophagy was studied (Fig. 1, I and J). Treatment of A549 cells with the mTOR inhibitor rapamycin (50 ng/ml) (39) resulted in an increased expression of LC3-II protein, and overexpression of HSP70 inhibited this up-regulation of LC3-II, indicating that HSP70 inhibits autophagy induced by rapamycin. Although in some experiments HSP70 overexpression seemed to affect the levels of the LC3-I band, the nature of the phenomenon has not been the focus of this study. We then tested whether rapamycin under control or starvation conditions affects HSP70 protein expression. To test this, A549 cells were treated with rapamycin (50 ng/ml) under control or starvation conditions (EBSS for 2 h). We found that neither rapamycin alone nor rapamycin under starvation conditions influenced HSP70 protein expression (supplemental Fig. S1).

p62 is a ubiquitously expressed cellular protein that serves as a common adapter for autophagy substrates and delivers them to autophagosomes where p62 along with the substrates is degraded (42–44). In the following experiments, we tested whether HSP70 will prevent p62 degradation. A549 cells were infected with either control (AdTrack) or HSP70 overexpressing virus for 72 h. Cells were either harvested, lysed, and the autophagy substrate protein p62 determined by Western blot analysis (Fig. 2, A and B) or immunostained, and p62 formation was imaged using Cellomics Array Scan (Fig. 2, C and D). Under full media conditions, overexpression of HSP70 resulted in both a significant increase in p62 protein expression determined by Western blot analysis and p62 formation determined by immunostaining. Starvation resulted in degradation of p62, and HSP70 overexpression significantly prevented this p62 degradation. These data along with the LC3 protein expression results (Fig. 1) demonstrated that HSP70 regulates autophagy in a cell culture model. To further investigate the intracellular mechanisms involved, the effect of starvation and HSP70 interaction on p62 mRNA levels was also examined (Fig. 2E). p62 mRNA expression did not show significant change, indicating that p62 is not regulated at the mRNA level under starvation and HSP70 overexpression conditions, and the observed changes in p62 represent post-translational modifications.

Effect of HSF-1 and High Temperature on Autophagy Regulation

The nuclear transcription factor HSF-1 has been shown to play an important role in regulating the expression of heat shock protein (45, 46). In the following experiment, we tested the hypothesis that HSF-1 plays an important role in autophagy regulation. HSF-1 expression was selectively knocked down by HSF-1 siRNA in A549 cells, and its role on autophagy regulation was studied. As shown in Fig. 3A, transfection of A549 cells with HSF-1 siRNA resulted in a marked depletion of HSF-1 expression. Under control conditions (full media with 100 nm Baf), siRNA depletion of HSF-1 resulted in a marked increase in autophagy, and HSF-1 knockdown potentiated starvation (EBSS with 100 nm Baf)-induced autophagy as demonstrated by an increase in LC3-II protein expression. These results suggest an important role of HSF-1 in regulating autophagy under both control and starvation conditions. In the following experiments, the effect of heat stress on starvation-induced autophagy was tested. A549 cells were initially exposed to heat exposure (42 °C for 2 h) followed by incubation at 37 °C for 24 h. Half of the cells were exposed to EBSS to induce autophagy under the presence of Baf (100 nm), and the other half was kept in full medium with Baf (100 nm). Cells were harvested, and HSP70, LC-3, and β-actin protein expression was determined by Western blot analysis. Heat stress exposure resulted in a significant increase in HSP70 in A549 cells (Fig. 3C). Interestingly, heat exposure resulted in a small but significant increase in autophagy as indicated by the increase in LC3-II protein expression (Fig. 3, C and D). Heat pre-conditioning in A549 cells exposed to EBSS resulted in an even larger increase in LC3-II protein expression, indicating that heat pre-conditioning potentiates starvation-induced autophagy. Next, the effect of HSF-1 on acute heat stress-induced autophagy in A549 cells was studied. First, A549 cells were transfected with HSF-1 siRNA for 48 h, followed by heat stress exposure (42 °C) for 2 h under Baf conditions. Immediately after acute heat exposure, cells were harvested, and Western blot analysis was performed. Exposure of A549 cells to HSF-1 siRNA under control temperature conditions (37 °C) led to an increase in autophagy (Fig. 3, E and F). Acute heat exposure did not result in an increase in autophagy. Cells exposed to the acute heat under HSF-1 inhibition resulted in a small autophagy inhibition compared with the HSF1 siRNA-treated cells and control temperature conditions. We also tested whether siRNA depletion of HSF-1 affected the unfolded protein response by measuring its indicators, including CHOP, XBP-1, and eIF2α (47). HSF-1 knockdown did not influence the unfolded protein response (supplemental Fig. S2). As a positive control, we utilized two commonly used unfolded protein response inducers thapsigargin (intracellular calcium releaser) (48) or tunicamycin (glycosylation inhibitor) (47). Exposure to thapsigargin (0.25 μm for 6 h) or tunicamycin (5 μg/ml for 24 h) resulted in a significant increase in phosphorylation of eIF2α and increased protein expression of XBP1 and CHOP (supplemental Fig. S2).

Effect of HSF-1 and HSP70 on Autophagy Regulation

Next, the role of HSF-1 and HSP70 on autophagy was examined. A549 cells were transfected with HSF-1 siRNA to knock down the HSF-1 protein expression followed by infection with adenovirus to overexpress HSP70 protein expression, and their role on starvation- and rapamycin-induced autophagy was tested. As shown in Fig. 4A, HSF-1 siRNA transfection resulted in a marked depletion of HSF-1 expression, and adenovirus infection with Ad70 resulted in a significant increase in HSP70 protein expression in A549 cells. No significant effects of HSP70 overexpression on HSF-1 protein levels were observed. As shown in Fig. 4, A and B, exposure to starvation conditions (EBSS with 100 nm Baf for 2 h) in A549 cells resulted in an increase in autophagy that was inhibited by HSP70 overexpression. Transfection with HSF-1 siRNA potentiated the starvation-induced increase in autophagy, and HSP70 significantly blocked this increase, indicating the strong regulatory role of HSP70 on autophagy. Similarly, transfection with HSF-1 siRNA potentiated the rapamycin-induced increase in autophagy that was prevented by HSP70 overexpression (Fig. 4, C and D).

In the following series of experiments, we further substantiated the regulatory role of HSP70 on macroautophagy in A549 cells by utilizing the recombinant wild type HSP70 and its ATPase domain mutant (K71A). A549 cells were either infected with control (AdTrack), HSP70 overexpressing virus (Ad70), or HSP70 mutant (Ad70M). As shown in Fig. 5, exposure to starvation conditions (EBSS with 100 nm Baf for 2 h) resulted in a significant increase in autophagy in A549 cells. HSP70 overexpression resulted in significant inhibition of this starvation-induced autophagy (Fig. 5, A and F). HSP70 ATPase mutant showed no significant effect on starvation-induced autophagy compared with that observed in starved cells infected with a control HSP70 virus, indicating that HSP70 ATPase domain is important in autophagy regulation.

It has been previously shown that the unfolded protein response triggered by the accumulation of misfolded protein in the ER induces autophagy (49) (50). One potential explanation of our results is that the HSP70-induced inhibition of autophagy was mediated by chaperone activity of HSP70. To test this hypothesis, A549 cells were transfected with pGL.3 to direct a high level of luciferase expression (51) along with control (AdTrack), HSP70 (Ad70), or HSP70 mutant (Ad70M) overexpressing virus. To denature the luciferase, cells were heated (45 °C for 45 min followed by a 4-h recovery period at 37 °C) and harvested, and the luciferase activity was determined. In cells overexpressing HSP70, the luciferase activity was over 2-fold higher than it was in control virus-infected cells, indicating that HSP70 prevented the luciferase activity (Fig. 5J). However, Ad70M did not render that protection, suggesting that HSP70 regulatory role of autophagy is mediated via its chaperone activity.

mTOR signaling plays a crucial role in numerous cellular processes, including cell growth, proliferation, and cell survival (52–55). The kinase mTOR is a main controller of autophagy induction, with inhibited mTOR activating the autophagy (56). We tested mTOR activation by monitoring mTOR phosphorylation (hypophosphorylation of mTOR indicates mTOR inhibition). Exposure of A549 cells to starvation conditions (EBSS with 100 nm Baf for 2 h) resulted in a hypophosphorylation of mTOR (Fig. 5, A and B) that was prevented by overexpression of HSP70 but not its mutant (Fig. 5, A and B). We also studied TOR activity by monitoring phosphorylation of eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) (Fig. 5, A and E), commonly used as an indicator of TOR activation (57, 58). Two isoforms of 4E-BP1 (β and γ) were highly phosphorylated in control cells. Starvation conditions resulted in an increase in the amount of the slowest migrating electrophoretic isoforms (α and β) of 4E-BP1, most probably as a result of hyperphosphorylation. This hyperphosphorylation of the two lowest bands disappeared in cells treated with Ad70. However, in starved cells treated with HSP70 ATPase mutant, the two lowest bands became apparent. Similarly, starvation resulted in a significant decrease in p70S6K phosphorylation (Fig. 5, A and C) that was partially prevented by HSP70 overexpression but not the HSP70 mutant. Considering its important role in autophagy regulation, we hypothesized Akt via activating mTOR (59) to be a target of HSP70 in autophagy regulation. To validate the role of HSP70 on the Akt pathway in autophagy regulation, A549 cells were infected with either control, HSP70 overexpressing virus, or its mutant (Ad70M), and the role of HSP70 on starvation-induced Akt activation was studied. As shown in Fig. 5, A and D, overexpression of HSP70, but not HSP70 ATPase mutant, under both control and starvation conditions, resulted in an increased phosphorylation of Akt, suggesting that HSP70 modulates autophagy through the Akt and mTOR pathway. In the following studies, the role of Akt in HSP70-mediated regulation of starvation-induced autophagy was studied (Fig. 5, G and I). Cells were treated with a highly selective inhibitor of Akt (3 μm MK-2206, Selleck Chemicals, Houston, TX) (60) and HSP70 overexpressing adenovirus (Ad70) (2.5 × 10¹⁰ IFUs) for 3 days. Subsequently, cells were exposed to EBSS (with 100 nm Baf) for 2 h. Cells were harvested, lysed, and prepared for Western blot analysis to measure the relative expression of LC3. In cells overexpressing HSP70, we observed an increase in mTOR and Akt phosphorylation that was associated with a decrease in starvation-induced autophagy. Treatment with an Akt inhibitor suppressed mTOR phosphorylation by 30 and 37% under the presence or absence of HSP70 adenovirus exposure, respectively. Exposure to selective Akt inhibitor resulted in a significant increase in autophagy that was not affected by HSP70, indicating that HSP70 exerts its inhibitory effect on autophagy via the Akt pathway.

Under control conditions, autophagy is responsible for the degradation of misfolded and unfolded proteins. Under stress conditions, cells utilize autophagy as a part of homeostatic mechanisms involved in maintaining sufficient macromolecular and ATP production. Although the role of exercise as a stimulus has been recently described in mice (35), the role of exercise on autophagy regulation in humans is less known. In this study, we sought to test the effect of exercise on autophagy regulation in humans under control conditions or glutamine supplementation. Exercise resulted in a significant increase in autophagy in human PBMCs as demonstrated by an increased LC3-II protein expression (Fig. 6, A and B). Gln treatment associated with a significant increase in HSP70 protein expression prevented this exercise-induced increase in autophagy.