Autophagy response to acute high-intensity interval training and moderate-intensity continuous training is dissimilar in skeletal muscle and peripheral blood mononuclear cells and is influenced by sex

Abstract

Autophagy is an evolutionary conserved cellular degradation system that underlies the positive effects of exercise. Currently, few human data exist investigating the autophagic response to exercise including the response to high-intensity interval training (HIIT), response in divergent tissues, and if sex differences exist. The purpose of this study was to investigate the autophagy response in skeletal muscle and peripheral blood mononuclear cells (PBMCs) following an acute bout of HIIT and moderate-intensity continuous training (MICT) with treadmill running in males and females. Using a crossover design, ten recreationally-active males (n=5; 25.2±1.1 yrs) and females (n=5; 21.6±3.6 yrs) performed a bout of MICT (60 minutes at 55% of max velocity [Vmax]]) and HIIT (12 bouts of 1 minute at 100% Vmax and 1 minute at 3 miles per hour) in a fasted state separated by ≥72 hours. Muscle biopsy samples from the vastus lateralis and PBMCs were collected pre- and 3 hours post-exercise and analyzed for differences in protein expression of LC3I, LC3II, and p62 via western blot analysis. Expression of LC3II:LC3I was significantly different from pre-exercise 3 hours post-exercise in MICT in skeletal muscle (64.3±47.3%; p=0.024). A significant time effect was found for p62 3 hours post-exercise compared to pre-exercise (135.23±84.6%; p=0.043) in skeletal muscle. No differences in markers of autophagy were observed in PBMCs. When sexes were analyzed separately there was a condition x time x sex interaction in LC3II (p=0.007) and LC3II:LC3I (p=0.043) in PBMCs. Post hoc analyses revealed a difference in LC3II pre vs. 3 hours post exercise in males, but not females, in both HIIT (144.2±89.7%; p=0.024) and MICT (61.8±36.1%; p=0.043). Our findings show that HIIT results in changes in markers of autophagy and that the exercise-induced autophagy response varies in tissues and between sexes.

Full text

23 (2021) 200118
Available online 26 December 2020
2666-1497/© 2020 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Autophagy response to acute high-intensity interval training and
moderate-intensity continuous training is dissimilar in skeletal muscle and
peripheral blood mononuclear cells and is inuenced by sex
Kurt A. Escobar
a
,
*
, Anna M. Welch
b
,
c
, Andrew Wells
b
, Zac Fennel
b
, Roberto Nava
b
, Zidong Li
b
,
Terence A. Moriarty
d
, Carlos H. Nitta
g
, Micah N. Zuhl
e
, Trisha A. VanDusseldorp
f
, Christine
M. Mermier
b
, Fabiano T. Amorim
b
a
Physiology of Exercise and Sport Lab, Department of Kinesiology, California State University, Long Beach, USA
b
Department of Health, Exercise & Sport Sciences, University of New, Mexico
c
Division of Occupational Therapy, University of New, Mexico
d
Department of Kinesiology, University of Northern Iowa, USA
e
School of Health Sciences, Central Michigan University, USA
f
Department of Exercise Science & Sports Management, Kennesaw State University, USA
g
Clinical Pathology Lab, Lovelace Biomedical Research Institute, USA
ARTICLE INFO
Keywords:
Autophagy
High-intensity interval training
Skeletal muscle
Peripheral blood mononuclear cells
Sex differences
Exercise
ABSTRACT
Autophagy is an evolutionary conserved cellular degradation system that underlies the positive effects of exer-
cise. Currently, few human data exist investigating the autophagic response to exercise including the response to
high-intensity interval training (HIIT), response in divergent tissues, and if sex differences exist. The purpose of
this study was to investigate the autophagy response in skeletal muscle and peripheral blood mononuclear cells
(PBMCs) following an acute bout of HIIT and moderate-intensity continuous training (MICT) with treadmill
running in males and females. Using a crossover design, ten recreationally-active males (n =5; 25.2 ±1.1 yrs)
and females (n =5; 21.6 ±3.6 yrs) performed a bout of MICT (60 min at 55% of max velocity [V
max
]]) and HIIT
(12 bouts of 1 min at 100% V
max
and 1 min at 3 miles per hour) in a fasted state separated by ≥72 h. Muscle
biopsy samples from the vastus lateralis and PBMCs were collected pre- and 3 h post-exercise and analyzed for
differences in protein expression of LC3I, LC3II, and p62 via western blot analysis. Expression of LC3II:LC3I was
signicantly different from pre-exercise 3 h post-exercise in MICT in skeletal muscle (64.3 ±47.3%; p =0.024).
A signicant time effect was found for p62 3 h post-exercise compared to pre-exercise (135.23 ±84.6%; p =
0.043) in skeletal muscle. No differences in markers of autophagy were observed in PBMCs. When sexes were
analyzed separately there was a condition x time x sex interaction in LC3II (p =0.007) and LC3II:LC3I (p =
0.043) in PBMCs. Post hoc analyses revealed a difference in LC3II pre vs. 3 h post exercise in males, but not
females, in both HIIT (144.2 ±89.7%; p =0.024) and MICT (61.8 ±36.1%; p =0.043). Our ndings show that
HIIT results in changes in markers of autophagy and that the exercise-induced autophagy response varies in
tissues and between sexes.
1. Introduction
Autophagy is an evolutionary conserved cellular recycling system
implicated in the adaptive responses and outcomes of exercise [1,2].
Autophagy maintains the intracellular environment through the degra-
dation of damaged and dysfunctional proteins, organelles, and lipids and
is associated with improved function [3,4]. Compromised autophagy
results in the accumulation of deleterious cytosolic components and
* Corresponding author. 1250 Bellower Blvd, HHS2-212, Long Beach, 90840, CA, USA.
E-mail addresses: kurt.escobar@csulb.edu, kurt.escobar@csulb.edu (K.A. Escobar), welcha@unm.edu (A.M. Welch), anwells@unm.edu (A. Wells), zfennel@unm.
edu (Z. Fennel), rnavabjj@unm.edu (R. Nava), zidongli1991@unm.edu (Z. Li), terence.moriarty@uni.edu (T.A. Moriarty), cnitta@lovelacebiomedical.org
(C.H. Nitta), zuhl1m@cmich.edu (M.N. Zuhl), tvanduss@kennesaw.edu (T.A. VanDusseldorp), cmermier@unm.edu (C.M. Mermier), amorim@unm.edu
(F.T. Amorim).
Contents lists available at ScienceDirect
Human Nutrition & Metabolism
journal homepage: www.sciencedirect.com/journal/human-nutrition-and-metabolism
https://doi.org/10.1016/j.hnm.2020.200118
Received 18 June 2020; Received in revised form 14 November 2020; Accepted 22 December 2020

Human Nutrition & Metabolism 23 (2021) 200118
2
contributes to tissue and organismal degeneration including numerous
chronic diseases [5–7]. Energetic stress including acute exercise stimu-
lates autophagy whereby cytosolic materials are engulfed by
double-membraned vesicles (autophagosomes) and transported to the
lysosome where they are degraded [8]. Degraded materials are released
back into the cytoplasm for energy and protein metabolism [3]. In
response to acute exercise, autophagy functions to provision substrates
for energy production and remains elevated hours after the cessation of
exercise [8–11]. Increasing evidences suggest autophagy plays a central
role in the adaptations and benecial effects of exercise [1,2,12]. Work
in animal models have shown autophagy is necessary for enhanced
endurance [13], mitochondrial biogenesis [13–15], angiogenesis [13],
and tissue remodeling in skeletal muscle [16] resulting from training.
Moreover, exercise stimulates autophagy in numerous tissues indicating
a global response [17–19]. However currently, scarce human data exist
describing the autophagic response to acute exercise including
comparing different exercise intensities and types as well as tissues.
High intensity interval training (HIIT) is an increasingly practiced
training method amongst active populations. HIIT exercise is charac-
terized by repeated bouts of brief (~30 s–4 min) high intensity (>80%
VO
2max
, HR
max
, peak power) exercise followed by periods of rest or low
intensity exercise (~30 s–4 min) that result in shorter training durations
compared to moderate-intensity continuous training (MICT) [20,21].
Several studies demonstrate that HIIT exercise can elicit equal or su-
perior physiological and health changes as traditional MICT when
amounts of work are similar or even lower [20–22]. Autophagy has been
shown to be upregulated following MICT exercise [23,24], however
currently, the autophagy response to HIIT exercise (in humans) is
unknown.
Acute exercise has been shown to induce autophagy in a number of
tissues including brain, heart, adipose tissue, pancreatic β cells [17],
liver [18], skeletal muscle [10,24,25], and peripheral blood mono-
nuclear cells (PBMCs) [19] suggesting an acute global response. This
widespread autophagic response may underlie the positive effects ex-
ercise elicits in numerous tissues [26]. In humans however, autophagy
activity in response to acute exercise in multiple tissues (i.e. skeletal
muscle and PBMCs) is not known. Additionally, sex differences in basal
as well as stress-induced autophagy have been reported between males
and females which varies between tissues [27–31]. However, few
human data exist investigating sex differences in autophagy levels
including in response to acute exercise.
The present study aimed to investigate the autophagy response to
acute HIIT and MICT exercise in skeletal muscle and PBMCs and
examine the inuence of sex therein. It was hypothesized both HIIT and
MICT would stimulate autophagy in skeletal muscle and PBMCs and that
this response would be different between males and females.
2. Materials and methods
2.1. Participants
Ten healthy, active young adult-aged males (n =5; age 25.2 ±1.1 yr,
height 180.2 ±6.4 cm, body mass 78.8 ±10.6 kg; VO
2max
: 48.0 ±4.9
ml/kg/min) and females (n =5; age 21.6 ±3.6 yr, height 162.5 ±11.7
cm, body mass 58.8 ±11.7 kg; VO
2max
: 39.4 ±7.7 ml/kg/min; Table 1)
who have been engaging in regular physical activity (>150 min of
moderate to vigorous intensity aerobic activity per week for a minimum
of 1 year) were recruited for the study. The study was approved by the
University of New Mexico Institutional Review Board (IRB). Participants
completed a health history questionnaire and a physical activity history
questionnaire to determine training and health status. Participants were
excluded if they were smokers, currently diagnosed with any disease,
currently taking any medications known to impact muscle metabolism
(e.g., statins, COX inhibitors, AMPK activators, etc.).
All values are mean ±SD. Yrs =years, cm =centimeters, kg =ki-
lograms, ml/kg/min =milliliters per kilogram per minute. *signies
signicant difference compared to males (p <0.05).
2.2. Experimental overview
The study required three visits to the laboratory; one visit for
screening and VO
2peak
determination followed by two randomized
experimental visits (HIIT and MICT). All visits were separated by at least
72 h. Experimental exercise bouts (HIIT and MICT) were performed in
the fasted state (≥8 h overnight fast). Muscle biopsies from the m. vastus
lateralis and venous blood draws were performed pre and 3 h post-
exercise following HIIT and MICT. Muscle biopsies were performed
using ne needle microbiopsy. Whole blood was processed for isolation
of PBMCs. Subjects remained fasted and were prohibited from
consuming caffeine or energy-containing beverages during the 3-h post-
exercise period.
2.3. Exercise testing and screening
Subjects arrived at the laboratory after abstaining from alcohol for
24 h, exercise for 24 h, and caffeine for 8 h for visit 1. This visit included
lling out health/exercise history and consent forms, a measurement of
resting blood pressure, 3 site skinfold body composition measurement,
and a maximal graded exercise test on a treadmill. The maximal graded
exercise test was used to assess VO
2peak
, as well as maximal velocity
(V
max
) which was used to determine running speeds for HIIT (100%
V
max
) and MICT (55% V
max
) exercise. High intensity interval training
and MICT exercises were performed at a 3% grade, therefore the graded
maximal exercise test was performed at 3% through the entirety of the
test. The test was individually designed using a valid equation estab-
lished by Jurca et al., 2005 [32] to estimate VO
2max
. Using the ACSM
running metabolic equation [33], estimated VO
2max
was used to deter-
mine estimated V
max
, which was then divided by 10 min to determine an
individualized speed increment per stage for the 10-min maximal graded
exercise test. Subjects warmed up for 3 min at 3 mph at 3% grade. Ex-
ercise started at minute 3 at 4 mph and 3% grade, and speed was
increased by the determined value every minute. During the maximal
graded running exercise test, heart rate was measured. Maximal speed
achieved during nal stage of the graded exercise test was used as V
max
in HIIT exercise and to determine MICT speed. Expired gases were
collected via a metabolic gas analyzer (Parvomedics, TrueOne 2400,
Sandy, UT) to assess VO
2peak
. Termination of the maximal graded ex-
ercise test occurred upon the subject reaching maximal exertion and
volitional cessation. Maximum oxygen consumption was determined
using 11-breath averaging. Not all subjects reached all three VO
2max
criteria (plateau of 150 ml/min, RER of 1.10, HR within ±10 bpm of
age-predicted HRmax), thus VO
2peak
was reported.
2.4. Exercise protocols
The HIIT exercise consisted of a 2-min warmup at 5 mph followed by
6 bouts of 1 min at V
max
obtained at VO
2peak
at 3% grade and 1 min of 3
mph and 3% grade. Subjects then had a 5-min rest period of walking 3
mph and 3% grade prior to another set of 6 bouts of 1 min at 100% of
Vmax at 3% grade and 1 min at 3 mph. This was followed by 2 min of
cooldown at 3 mph. The MICT bout consisted of a 2-min warm up at 5
mph, 60 min at 55% Vmax and 3% grade, and 2 min of cooldown at 3
Table 1
Subject characteristics.
Males (n =5) Females (n =5) p-value
Age (yrs) 25.2 ±1.1 21.6 ±3.6 0.07
Height (cm) 180.2 ±6.4 162.5 ±11.7* <0.01
Body Mass (kg) 78.8 ±10.6 58.8 ±11.7* 0.02
Body Fat (%) 9.2 ±2.6 18.1 ±5.0* <0.01
VO
2max
(ml/kg/min) 46.0 ±4.3 35.1 ±7.7* 0.02
K.A. Escobar et al.

Human Nutrition & Metabolism 23 (2021) 200118
3
mph. Including the warm-up and cooldown, the HIIT bout was 31 min in
duration; the MICT bout was 64 min in duration. If subjects could not
sustain the speed of the HIIT (V
max
bout) or MICT protocols, speed was
reduced by (to the nearest) 10% in order to complete the exercise bout.
Heart rate was measured throughout both HIIT and MICT exercise bouts.
2.5. Skeletal muscle biopsy
Muscle was extracted from the lateral portion of the m. vastus lateralis
midway between the patella and the iliac crest using the ne needle
microbiopsy technique pre- and 3 h post-exercise. The self-reported
dominant leg was used for all biopsies. After hypodermic administra-
tion of 2% Lidocaine, the leg was punctured using an 18-gauge pilot
needle to a depth of 5–10 mm. A 14-gauge biopsy needle was loaded into
the spring-loaded microbiopsy instrument (Pro-Mag
TM
Ultra Automatic
Biopsy Instrument; Argon Medical Devices, Frisco, TX, USA) and inser-
ted into the leg via the pilot incision to extract skeletal muscle tissue. The
muscle sample was obtained by the activation of a trigger button that
unloaded the spring and activated the needle to collect muscle tissue
(~10 mg). The biopsy needle was removed from the pilot hole and the
tissue sample collected. The biopsy needle was then reloaded in the
biopsy instrument and reinserted into the pilot incision for an additional
sample collection. All tissue samples were immediately frozen in liquid
nitrogen and stored in a −80 ◦C freezer for subsequent analysis.
2.6. PBMC isolation
Approximately 12 ml of venous blood was collected from the median
cubital vein pre-exercise and 3 h post-exercise in ethyl-
enediaminetetraacetic acid (EDTA)-treated tubes for the isolation of
PBMCs. Whole blood was transferred onto 10 ml of histopaque-1077
(Sigma Aldrich, St. Louis, MO, USA), and centrifuged at 2400 rpm for
30 min. The PBMC layer was pipetted into 10 ml of RPMI-1640 Medium
(Sigma Aldrich). Cells were then washed using phosphate buffered sa-
line (PBS; Sigma Aldrich) and centrifuged at 13,000 rpm for 10 min at
two times at 4 ◦C in a microcentrifuge before being aliquoted into
Eppendorf tubes and frozen at −80 ◦C until analysis.
2.7. Protein quantication
Skeletal muscle was homogenized in 1 ml Lysis Buffer 3 (Cloud
Clone, Katy, TX) per 50 mg of tissue using BeadBug zirconium prelled
tubes (Sigma Aldrich, St. Louise, MO). Muscle tissue was spun in a
BeadBug microtube homogenizer (Sigma Aldrich, St. Louise, MO) twice
for 40 s at 4000 rpm. Samples were then spun for 2 min at 13,000 rpm at
4 ◦C in a microcentrifuge. Supernatant was then collected and stored for
protein quantication. Supernatant was further diluted 1:20 with Cloud
Clone Lysis Buffer 3 for protein quantication which was performed
using Pierce BCA Protein Assay Kit (Thermo Fisher Scientic, Waltham,
MA).
PBMCs were lysed in a modied RIPA buffer (Tris-HCl 8.0 pH;
Invitrogen, Carlsbad, CA, USA); 0.5 M EDTA (Invitrogen), 1.5 M NaCl
(Sigma-Aldrich) 1% Triton X 100 (Sigma-Aldrich), and freshly added
protease (ThermoScientic, Carlsbad, CA, USA) and phosphatase in-
hibitors (ThermoScientic).
A 2X Laemmli buffer with 5% β-mercaptoethanol was added to the
protein lysates and incubated at 100 ◦C for 10 min before gel loading for
protein separation by electrophoresis. Fifteen ug of protein per sample
was loaded into a resolving and stacking sodium dodecyl sulfate poly-
acrylamide gel. Separated proteins were transferred to a Trans-blot
Turbo transfer PVDF membrane (Bio-Rad Laboratories, Hercules, CA,
USA). Membranes were blocked for 30 min in 5% dry milk and Tris
buffered saline Tween 20 buffer solution, washed in Tris-buffered saline
(TBS), and incubated overnight in the LC3 (Sigma Aldrich, St. Louise,
MO) and p62 (Cell Signaling Technology, Danvers, MA) antibodies at
4 ◦C. All membranes were washed in TBS with 0.05% Tween 20 (TBS-
tween) and incubated with a horseradish peroxidase-conjugated sec-
ondary antibody (Invitrogen, Carlsbad, CA) and incubated for 1 h at
room temperature. Western Blotting Luminol Reagent (Santa Cruz
Biotechnology, Santa Cruz, CA) using the ChemiDoc Touch Imaging
System (Bio-Rad, Hercules, CA) was used to develop and record the
membrane. Image Lab software (Bio-Rad, Hercules, CA) was used to
quantify protein expression by determining densitometric values. All
proteins were normalized to total protein loaded.
2.8. Statistical analysis
Mean differences in LC3I, LC3II, LC3II:LC3I, and p62 were assessed
between and HIIT and MICT exercise using a 2 (HIIT and MICT) x 2 (pre-
exercise and 3 h post-exercise) repeated measures ANOVA in skeletal
muscle and in PBMCs. Mean differences in markers of autophagy be-
tween males and females were also assessed between HIIT and MICT in
skeletal muscle and PBMCs using a 2 (HIIT and MICT) x 4 (pre-exercise
males, pre-exercise females, 3 h post-exercise males, and 3 h post-
exercise females) repeated measures ANOVA. Three hours post-
exercise values were compared to pre-exercise which was given a stan-
dard value of 100%. When appropriate, a Bonferroni post-hoc test was
completed. A student’s T Test was used to determine mean differences in
HR between HIIT and MICT. A power analysis based on previous work
[34] determined a sample size of 10 subjects was adequate for both
combined and separated sex analyses. All statistics were done using SPSS
(Version 25). The threshold for statistical signicance was set a priori at
a p-value of ≤0.05 for all tests.
3. Results
All subject completely performed HIIT and MICT protocols with only
several instances of reducing running speed. Mean HR was signicantly
different (p <0.01) between HIIT (159 ±14 bpm) and MICT (154 ±8
bpm; Fig. 1). No differences were detected in LC3I or LC3II protein
content 3 h post-exercise compared to pre-exercise in either HIIT and
MICT bouts in skeletal muscle or PBMCs (p >0.05). However, there was
a time x condition interaction for LC3II:LC3I ratio (p =0.046). A post
hoc analysis revealed LC3II:LC3I was signicantly lower at 3 h post-
exercise (64.3 ±47.3%) compared to pre-exercise in skeletal muscle
(p =0.024; Fig. 2). There was also a main effect for time for p62
expression pre vs. 3 h post-exercise (135.23 ±84.6%) in skeletal muscle
(p =0.043; Fig. 3). There were no differences in LC3II:LC3I or p62 in
PBMCs between HIIT and MICT pre vs. 3 h post-exercise (p >0.05).
When sexes were analyzed separately, there was a time x condition x
sex interaction for LC3II (p =0.007) and LC3II:LC3I (p =0.043) in
Fig. 1. Mean heart rate (HR; bpm) during high-intensity interval training
(HIIT; black line) and moderate-intensity continuous training (MICT; grey line)
in physically active males (n =5) and females (n =5). * signies signicant
difference between HIIT and MICT (p <0.01).
K.A. Escobar et al.

Human Nutrition & Metabolism 23 (2021) 200118
4
PBMCs, but no sex interaction effects in skeletal muscle. A post hoc
analysis revealed a difference in LC3II between pre-exercise and 3 h
post-exercise in males in HIIT (144.2 ±89.7%; p =0.024) and MICT
(61.8 ±36.1%; p =0.043) in PBMCs (Fig. 4A). There was no difference
in LC3II in females. There was no signicant differences found in either
sex in HIIT or MICT in LC3II:LC3I in the post hoc analysis (Fig. 4B).
Fig. 2. Representative blots of A. LC3I and
LC3II protein expression, B. total protein,
and C. percent change (%) ratio from pre-
exercise (Pre) to 3 h post-exercise (3 h)
following post high-intensity interval
training (HIIT) and moderate-intensity
continuous training (MICT) in skeletal mus-
cle (solid bars) and peripheral blood mono-
nuclear cells (PBMCs; empty bars) of 10
physically active males (n =5) and females
(n =5). Quantication of relative protein
content was completed using densitometric
values obtained using Image Lab software
and normalized to total protein loaded and
set to 100% for pre-exercise. Bar graph rep-
resents means and standard error. *signies
signicant difference from Pre MICT in
skeletal muscle (p =0.02).
Fig. 3. Representative blots of A. p62 pro-
tein expression, B. total protein, and C.
percent change (%) from pre-exercise (Pre)
to 3 h post-exercise (3 h) following post
high-intensity interval training (HIIT) and
moderate-intensity continuous training
(MICT) in skeletal muscle (solid bars) and
peripheral blood mononuclear cells (PBMCs;
empty bars) of 10 physically active males (n
=5) and females (n =5). Quantication of
relative protein content was completed using
densitometric values obtained using Image
Lab software and normalized to total protein
loaded and set to 100% for pre-exercise. Bar
graph represents means and standard error.
+signies time effect in skeletal muscle (p
=0.04.
Fig. 4. Representative blots of A. LC3I and LC3II protein expression, B. total protein, and percent change (%) of C. LC3II:LC3I and D. LC3II from pre-exercise (Pre) to
3 h post-exercise (3 h) following post high-intensity interval training (HIIT) and moderate-intensity continuous training (MICT) in peripheral blood mononuclear cells
(PBMCs) of physically active males (n =5; solid bars) and females (n =5; empty bars). Quantication of relative protein content was completed using densitometric
values obtained using Image Lab software and normalized to total protein loaded and set to 100% for pre-exercise. Bar graph represents means and standard error. ^
signies time x condition x sex interaction in LC3II (p =0.007) and LC3II:LC3I (p =0.043); # signies difference between pre and 3 h post-exercise in males in HIIT
(p =0.024) and MICT (p =0.04) in LC3II.
K.A. Escobar et al.

Human Nutrition & Metabolism 23 (2021) 200118
5
There were no differences in LC3I or p62 in males or females. A uni-
variate ANOVA of pre-exercise LC3I, LC3II, LC3II:LC3I, and p62
revealed there were no signicant differences between males and fe-
males at rest.
4. Discussion
Autophagy is increasingly implicated in the adaptive responses to
exercise and appears to be activated in a duration and intensity
dependent manner [8]. However, human data are scarce and inconsis-
tent in characterizing thresholds of exercise to stimulate autophagy.
These are the rst data to the authors’ knowledge assessing autophagy
activity in skeletal muscle and PBMCs following acute HIIT and MICT
exercise and examining the inuence of sex therein. In our study we
show HIIT is capable of increasing autophagic ux however this
response is distinct in three ways: 1) MICT, but not HIIT, altered LC3II:
LC3I 3 h post-exercise in skeletal muscle, 2) p62 was increased in
response to both HIIT and MICT exercise in skeletal muscle, and 3) HIIT
and MICT altered autophagy in males, but not females in PBMCs.
Hight-intensity interval training is a popular form of exercise that
produces benecial outcomes similar to MICT [21]. The present inves-
tigation is the rst to study the autophagy response to HIIT. Three hours
following MICT, LC3II:LC3I signicantly decreased in skeletal muscle to
64.3 ±47.3% of pre-exercise while no change was observed in HIIT
(108.1 ±54.5%). A discrepant LC3II:LC3I response between HIIT and
MICT bouts is a novel nding. In previous work, LC3II:LC3I ratio has
been shown to decrease in skeletal muscle in response to MICT exercise,
similar to our results, while others have shown no difference. Moller
et al. [24] reported 60 min of cycling at ~50% VO
2max
resulted in a
decrease in LC3II:LC3I immediately post-exercise. Similarly, Schwalm
and colleagues [10] showed 2 h of cycling at 55% and 70% VO
2max
in a
fasted and fed state decreased LC3II:LC3I immediately post- and 1 h
post-exercise with the exception of 1 h after the 55% VO
2max
bout in the
fed state. Alternatively, cycling for 20 min at ~50% VO
2max
[35] and 60
min at ~70% VO
2max
[36] did not cause changes in LC3II:LC3I ratio;
there were no signicant changes in LC3I or LC3II in either study. These
data indicate the response of LC3 to exercise is variable. Our results
support this as well, noting decreased LC3II:LC3I following MICT, but
not in HIIT which might be related to duration of the exercise. However,
LC3 status (i.e. LC3I and LC3II) is dynamic and based on baseline
transcriptional activity (LC3I), lipidation (LC3I to LC3II), and autopha-
gasomal degradation (LC3II) during autophagic ux [37]. Thus, auto-
phagy activity may be represented by varying compositions in LC3 as
demonstrated by previous work (Jamart, Francaux, Millet, Deldicque,
Frere, et al., 2012; Schwalm et al., 2015). Secondary markers of auto-
phagic activity (i.e. p62) may be used to corroborate changes in auto-
phagic ux (Klionsky, Abdelmohsen et al., 2016).
The autophagy substrate, p62, is an adapter protein associated with
autophagasome formation and is used as an indicator of autophagic ux
[3]. Here we showed, similar to MICT, HIIT increases p62 in skeletal
muscle. Schwalm et al. [10] also reported increase p62 protein expres-
sion 1 h post-exercise in both 55% and 70% VO
2max
intensities in both
fasted and fed states. The increase in p62 with exercise may indicate an
increase in autophagic ux. p62 is degraded with the autophagasome at
the lysosome and its upregulation is usually associated with periods of
elevated clearance after an initial decrease [38]. Thus, an increase in
p62 following acute exercise may not be unexpected and suggests
elevated transcription observed at 3 h post-exercise.
This study is also the rst to assess autophagy activity following an
acute bout of exercise in skeletal muscle and PBMCs. While LC3II:LC3I
and p62 were affected in skeletal muscle, no markers of autophagy were
affected in PBMCs when all subjects (male and female) subjects were
analyzed together. Previous works show acute exercise stimulates
autophagy in PBMCs [9]. For example, cycling at 50% VO
2max
for 60 min
with 5 min of rest interspersed every 20 min increased markers of
autophagy immediately post-exercise in sedentary adults [39].
Additionally, Dokladny and colleagues [19] showed that treadmill
running for 60 min at 70–80% of VO
2max
caused an increase in LC3II
protein expression immediately, 2, and 4 h post-exercise, however
running was completed in a warm environmental chamber (30 ◦C).
These, in combination with our data, may suggest different exercise
thresholds in specic tissues for autophagy activation that may be
affected by population and environmental stresses.
Sex differences in autophagy levels and gene expression (Atgs) have
been observed in animals in multiple tissues including in response to
stress [27–30]. However, very few human data exist investigating sex
differences in autophagy activity despite the role autophagy serves in
human function and health. To the authors’ knowledge no studies have
investigated differences in human males and females including in
response to acute exercise. In the present study sexes were separated and
analyzed to determine if the exercise-induced autophagy response was
affected by sex. At rest there were no differences between autophagy
markers in skeletal muscle or PBMCs. However, in PBMCs, there was a
signicant condition (HIIT and MICT) x time (pre-exercise and 3 h
post-exercise) x sex (male and female) interaction in LC3II (p =0.007)
and LC3II:LC3I. (p =0.043). Bonferroni post hoc analyses revealed
differences in LC3II 3 h post-exercise vs. pre-exercise in males, but not in
females, in both HIIT (144.2 ±89.7%; p =0.024) and MICT (61.8 ±
36.1%; p =0.043). No signicant differences were found in LC3II:LC3I
in the post hoc analysis between sexes in HIIT or MICT. There were no
interaction effects of sex in skeletal muscle. To the authors’ knowledge,
these are the rst data demonstrating a sex difference in
exercise-induced autophagy. Whereas Smiles et al. [40] reported no sex
interactions in response to acute resistance training, we observed an
interaction in PBMCs where autophagy activity was altered
post-exercise in males, but not in females in response to HIIT and MICT.
Phase of menstrual cycle and hormonal contraception status were not
controlled for or reported in the current study. This may have inuenced
the (lack of) autophagy response to acute exercise in females, in both
skeletal muscle and PBMCs. Estradiol, which uctuates through the
menstrual cycle and is varied with hormonal contraception, dependent
on type as well as dosaging (Sims & Heather, 2018), inhibits autophagy
via the mammalian target of rapamycin complex 1 (mTORC1) (Park
et al., 2016). These data are interesting as they depict a sex- and
tissue-distinct autophagic response to exercise. Further investigation is
warranted into characterizing sex differences in the exercise-autophagy
response which should consider menstrual phase and contraception
status in pre-menopausal females.
The present study possesses several limitations including limited
markers of autophagic activity measured, sample size, particularly when
sexes were separated, and limited time points used (pre- and 3 h post-
exercise). Additionally, as with all HIIT/MICT comparison work dis-
crepancies in workload between HIIT and MICT exist. Such limitations
should be considered in subsequent work.
5. Conclusion
Autophagy has emerged as a key player in health and aging, and
growing evidence support that it is a mediator in the benecial re-
sponses to exercise. However, the autophagy response to HIIT had not
been previously investigated. Our study shows HIIT increases autopha-
gic ux, however in a non-identical manner to MICT (i.e. LC3II:LC3I in
skeletal muscle) and varies between tissues and sex. Our data add to the
currently-sparse literature of exercise-induced autophagy in humans
and may suggest it is involved in the adaptive responses to HIIT. The
discrepancy in response between MICT and HIIT may be attributed to
differences in exercise duration although further research is needed. It
should be noted, however, that exercise and physical activity far below
the durations and intensities observed here and elsewhere that elicit an
autophagy response [10,25,41], such as walking, have been shown to
produce increases in basal autophagy and expression of Atgs alongside
the positive effects on human health and function known of exercise [42,
K.A. Escobar et al.

Human Nutrition & Metabolism 23 (2021) 200118
6
43]. Therefore, it is an interesting question whether acute increases in
autophagic ux in response to a single bout of exercise are necessary to
produce chronic changes in autophagy. Given the growing under-
standing of the implications of autophagy on human function and
longevity and the effects of exercise therein, additional work should be
conducted to characterize the dose- and type-response of exercise on
autophagy in different tissues as well as investigating sex differences.
Funding
This research was in part funded by the Graduate and Professional
Student Association of the University of New Mexico.
CRediT authorship contribution statement
Kurt A. Escobar: Conceptualization, Data curation, Formal analysis,
Funding acquisition, Investigation, Methodology, Project administra-
tion, Resources, Software, Supervision, Writing - original draft, Writing -
review & editing. Anna M. Welch: Data curation. Andrew Wells: Data
curation. Zac Fennel: Data curation. Roberto Nava: Formal analysis.
Zidong Li: Data curation. Terence A. Moriarty: Data curation. Carlos
H. Nitta: Formal analysis, Methodology. Micah N. Zuhl: Methodology,
Writing - review & editing. Trisha A. VanDusseldorp: Methodology,
Writing - review & editing. Christine M. Mermier: Conceptualization,
Data curation, Formal analysis, Methodology, Supervision, Writing -
review & editing. Fabiano T. Amorim: Conceptualization, Data cura-
tion, Formal analysis, Methodology, Supervision, Writing - review &
editing.
Declaration of competing interest
The authors declare that they have no known competing nancial
interests or personal relationships that could have appeared to inuence
the work reported in this paper.
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