Received: 1 October 2019 Revised: 13 December 2019 Accepted: 19 December 2019

DOI: 10.1002/dta.2755

SPECIAL ISSUE - RESEARCH ARTICLE

Beta2-adrenergic agonist clenbuterol increases energy

expenditure and fat oxidation, and induces mTOR
phosphorylation in skeletal muscle of young healthy men

Søren Jessen1 | Sara A. Solheim1 | Glenn A. Jacobson2 | Kasper Eibye1 |

1 1 1
Jens Bangsbo | Nikolai B Nordsborg | Morten Hostrup

1
Section of Integrative Physiology,
Department of Nutrition, Exercise and Sports,
University of Copenhagen, Denmark
2
School of Medicine, University of Tasmania,
Australia
Correspondence
Morten Hostrup, Section of Integrative
Physiology, Department of Nutrition, Exercise
and Sports, University of Copenhagen, August
Krogh Building 2nd floor, Universitetsparken
13, DK-2100 Copenhagen. Denmark.
Email: mhostrup@nexs.ku.dk
Funding information
Anti Doping Denmark, Grant/Award Number:
N/A
Abstract
Clenbuterol is a beta2-adrenoceptor agonist marketed as an asthma reliever but is
not approved for human use in most countries due to concerns of adverse cardiac
effects. Given its demonstrated hypertrophic and lipolytic actions in rodents,
clenbuterol is one of the most widely abused doping substances amongt athletes and
recreational body-builders seeking leanness. Herein, we examined the effect of
clenbuterol ingestion on metabolic rate as well as skeletal muscle mammalian target
of rapamycin (mTOR) phosphorylation and protein kinase A (PKA)-signaling in six
young men. Before and 140 min after ingestion of 80 μg clenbuterol, resting meta-
bolic rate and contractile function of the quadriceps muscle were measured, and
blood samples as well as vastus lateralis muscle biopsies were collected. Clenbuterol
increased resting energy expenditure by 21% (P < 0.001), and fat oxidation by 39%
(P = 0.006), whereas carbohydrate oxidation was unchanged. Phosphorylation of
mTORSer2448 and PKA substrates increased by 121% (P = 0.004) and 35%
(P = 0.006), respectively, with clenbuterol. Maximal voluntary contraction torque
decreased by 4% (P = 0.026) and the half-relaxation time shortened by 9%
(P = 0.046), while voluntary activation, time to peak twitch, and peak twitch torque
did not change significantly with clenbuterol. Glycogen content of the vastus lateralis
muscle did not change with clenbuterol. Clenbuterol increased circulating levels of
glucose (+30%; P < 0.001), lactate (+90%; P = 0.004), insulin (+130%; P = 0.009), and
fatty acids (+180%; P = 0.001). Collectively, these findings indicate that clenbuterol is
an efficient thermogenic substance that possibly also exerts muscle hypertrophic
actions in humans. For these reasons, the restrictions imposed against clenbuterol in
competitive sports seem warranted.

KEYWORDS

doping, wada, muscle hypertrophy, muscle growth, anabolic

1 | I N T RO DU CT I O N Clenbuterol was marketed in 1977 as an asthma reliever,2 but due to

concerns of adverse cardiac effects, the drug is no longer marketed
Clenbuterol is a beta2-adrenoceptor agonist (beta2-agonist), which for human use in most countries. Nevertheless, clenbuterol is one of
also possesses moderate affinity for beta1-adrenoceptors.1 the most heavily abused substances among bodybuilders and amateur

610 © 2019 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/dta Drug Test Anal. 2020;12:610–618.
JESSEN ET AL.
fitness athletes seeking leanness, as evidenced by numerous reports
of clenbuterol toxicity as a consequence of misuse for anabolic pur-
3-7
poses. Further highlighting its misuse for performance enhance-
ment, clenbuterol has accounted for more than 300 adverse analytical
findings (AAF) from routine doping control yearly since 2014.8
Clenbuterol is filed under “Anabolic Agents” in the S1-category
on the World Anti-Doping Agency (WADA)’s list of prohibited sub-
stances and methods, whereas other beta2-agonists appear in their
own category (the S3-category). Due to the classification of
clenbuterol as an anabolic agent, any amount of clenbuterol detected
in urine during doping control will result in an AAF. Exceptions to this
are cases where clenbuterol concentrations of <5 ng/mL are detected
in urine samples, which are classified as an “atypical finding”, and may
be traced back to meat contamination due to clenbuterol use in
livestock.9
In rodents, clenbuterol has been shown repeatedly to be a power-
ful anabolic and lipolytic substance,10-14 and on online bodybuilding
forums, clenbuterol is hailed for its purported ability to increase lean
mass while simultaneously decreasing fat mass. A progressive increase
in daily dose from 40 to 120 μg in 4–6 week on/off cycles seems to
be the most common strategy for doping with clenbuterol to purport-
edly reduce side effects.15 Despite the prohibition of clenbuterol
usage in sports and the evidence of the anabolic and lipolytic effects
of clenbuterol in rodents, no studies have investigated the physiologi-
cal effects of this drug with respect to its purported hypertrophic and
metabolic actions in healthy humans.
Here, we investigated the effect of clenbuterol ingestion on rest-
ing energy expenditure and fat oxidation, as well as on mTORSer2448
phosphorylation response in skeletal muscle in young men. We
hypothesized that clenbuterol would elevate resting energy expendi-
Ser2448
ture and fat oxidation, while inducing mTOR phosphorylation in
skeletal muscle.
2 | METHODS
2.1 | Subjects and ethical approval
Six male subjects were included in the study. Inclusion criteria were
healthy males aged 18–40 years with no known contraindications for
anabolic drugs as well as oral and written informed consent. Exclusion
criteria were self-reported steroid abuse, ongoing use of prescription
medicine, heavy resistance training more than 2 times weekly,
TABLE 1 Cardiovascular parameters and plasma K+ before (0 min) and after (140 min) ingestion of 80 μg clenbuterol
0 min
Heart rate (bpm) 51 (± 9)
Systolic blood pressure (mmHg) 115 (± 10)
Diastolic blood pressure (mmHg) 63 (± 5)
Mean arterial pressure (mmHg) 80 (± 5)
Plasma K+ concentration (mmol/L) 3.8 (± 0.1)
Values are mean (± SD) unless otherwise specified.
611
abnormal electrocardiography (ECG), or disease deemed by the study
responsible medical doctor to infer a risk to participate in the trial.
The study was conducted in accordance with the standards set by the
2013 version of the Declaration of Helsinki and was approved by
the regional research ethics committee of the Capital Region,
Denmark (H-17011319). The study was registered in clinicaltrials.gov
(NCT03860870).
The six subjects were (mean ± SD) 23 ± 5 years old, with a height
of 185 ± 6, and weight of 78 ± 8 kg.
The study is part of a larger research project aiming to develop
strategies to detect clenbuterol abuse for doping control purposes. In
the present part, outcome measures that pertain to the physiological
response to clenbuterol are presented.
2.2 | Study design and experimental procedures
The study was an open-label trial. Subjects arrived at the laboratory in
the morning after an overnight fast. The subjects rested in the supine
position for 30 min before the resting metabolic rate was determined
breath-by-breath by indirect calorimetry using a gas analyzer system
(Oxycon Pro, CareFusion, San Diego, CA, US) for 15 min. Blood pres-
sure was determined with an automatic sphygmomanometer
(M7 Intellisense, OMRON, Kyoto, Japan) on the left upper arm and a
blood sample was drawn in a heparinized syringe from a peripheral
venous catheter placed in the antecubital vein. Furthermore, a muscle
biopsy was collected from the vastus lateralis muscle of the left leg
using a Bergström needle with suction.16 Prior to biopsy sampling, an
incision (3 mm) was made through the skin and fascia at the vastus
lateralis belly under local anesthesia (2 mL lidocaine without epineph-
rine, 20 mg/mL Xylocain®, AstraZeneca, Cambridge, UK). Then, con-
tractile function of the quadriceps muscle of the right leg was
determined, before 80 μg oral clenbuterol (Spiropent, Boehringer
Ingelheim International GmbH, Ingelheim am Rhein, Germany) was
administered with water. This dose of clenbuterol was chosen based
on the commonly administered dose among recreational athletes
misusing clenbuterol for doping purposes.15 Subjects then rested in
the supine position for 140 min followed by the exact same proce-
dures as described above.
After the final measurements, subjects received a sandwich and
stayed at the laboratory for another 3 h after which blood pressure
and ECG were evaluated by a medical doctor. Apart from slightly
increased heart rate, no changes in ECG or blood pressure were
140 min Δ-change (95% CI) P-value
71 (± 17) 19 (10 to 29) 0.002
120 (± 9) 5 (−1 to 10) 0.068
61 (± 8) -2 (−9 to 5) 0.472
80 (± 8) 0 (−5 to 6) 0.944
3.5 (± 0.4) −0.3 (−0.6 to 0.1) 0.156
612
evident for any of the subjects. Then, subjects were asked if they had
experienced any side effects during the trial. A schematic representa-
tion of the study design is presented in Figure 1.
2.3 | Resting energy expenditure and substrate
oxidation
Energy expenditure and oxidation of fat and carbohydrates were
quantified using indirect calorimetry as previously described17 and
were calculated using the following equations deduced by
_ 2 + 1.1 × VCO
Ferranini:18 Energy expenditure = (3.91 × VO _ 2 )/1000–
3.34 × 0.14 × body weight/1440. Carbohydrate oxidation = ((4.55 ×
_
VCO 2 – 3.21 × VO _ 2 )/1000–2.87 × 0.14 × body weight/
_ 2 – 1.67 × VCO
1440) × 1000. Fat oxidation = ((1.67 × VO _ 2 )/1000–
1.92 × 0.14 × body weight/1440) × 1000. Energy expenditure is in
kcal/min, and fat and carbohydrate oxidation are in mg/min.
2.4 | Skeletal muscle biopsy preparation
Following collection, the muscle biopsy sample (~50–100 mg) was
washed in ice-cold saline to reduce blood contamination, and then
frozen in liquid nitrogen and stored in cryotubes at −80 C for later
Western blot analysis. Prior to this analysis, the biopsy samples were
freeze-dried and dissected free of visible fat, blood, and connective
tissue under a microscope.
2.5 | Blood analysis
After collection, venous blood samples were immediately analyzed in
a blood gas analyzer for determination of plasma K+, glucose, and lac-
tate (ABL 800 FLEX, Radiometer, Copenhagen, Denmark). For deter-
mination of plasma insulin and fatty acids, blood samples were
collected in Eppendorf tubes with 30 μL EDTA (0.2 M) and spun at
3000 rpm for 5 min, after which the plasma was collected and stored
at −80 C until analysis. Plasma insulin concentrations were deter-
mined for each subject with a commercially available Ultrasensitive
ELISA kit (Ref 80-INSHUU-E10, Alpco, NH, US) and analyzed on a
Multiskan FC microplate photometer (Thermo Fisher Scientific, MA,
US). Plasma fatty acid concentrations were determined for each sub-
ject with a commercially available NEFA-HR(2) kit (Fujifilm WACO
Chemicals, Neus, Germany) and analyzed on a Pentra C400 spectro-
photometer (Horiba ABX SAS, Montepellier, France).
JESSEN ET AL.
2.6 | Quadriceps contractile function
The contractile function of the quadriceps muscle was determined as
described previously.19 Subjects were seated on a customized chair
with their right leg secured to a strain gauge (Vishay Precision Group,
Tedea Huntleigh, model no. 615) by velcro tape placed just above the
malleoli. The right leg was visually adjusted to 90 flexion. Subjects
were instructed to hold on to metal bars secured to the table during
contraction. Prior to initiation of the test, self-adhesive electrodes
(PALS Platinum, Model 895240, Axelgaard Manufacturing Co., Ltd
DK, 8520 Lystrup, Denmark) were placed on the skin of the right
thigh above the rectus femoris and vastus lateralis muscles at 25%
and 75% of the distance measured between spina iliaca anterior supe-
rior and the base of the patella.
Each session consisted of a 3–4 s maximal voluntary contraction
under strong verbal encouragement with no visual feedback. When a
plateau in torque production was reached, an electro-stimulation was
delivered to create a superimposed twitch (Digitimer, model DS7AH,
Welwyn Garden City, England). Another electro-stimulation was
applied 1 s following relaxation to create a potentiated twitch. Three
sessions separated by 1 min of rest were conducted.
The strain gauge signal was received by an amplifier
(AD instruments, Octal bridge Amp, model ML228, Sydney, Australia)
connected to a computer. Data were recorded at 1 kHz and analyzed
in LabChart 7 (AD Instruments, v. 7.3.8, Sydney, Australia). The fol-
lowing parameters were determined: Maximal voluntary contraction
torque (MVC);Nm): highest torque during a maximal voluntary con-
traction; peak twitch torque (Nm): highest torque developed during
potentiated stimulation 1 s following relaxation from MVC; time-to-
peak twitch torque: time from potentiated stimulation to peak twitch
torque; half-relaxation time: time from peak twitch torque to half of
peak twitch torque. The distance from the base of patella to the mid-
dle of the velcro strap on the ankle was registered and used to calcu-
late torque in newton meters (Nm).
The highest MVC and peak twitch value, and the lowest half
relaxation time and time to peak twitch value from each time point
were used. Voluntary activation (VA) was calculated as described by
Bachasson et al.,20 using the following equation: VA = [1 – (TSI/
TPot)] × 100, where TSI is the superimposed twitch on top of MVC and
TPot is the potentiated twitch after relaxation. In the case where TSI
was delivered slightly before or after MVC, a correctional equation
was used as described by Strojnik and Komi:21 VA = [100 – (Tb –
MVCpeak) × (Tb/MVCpeak)/TSI] × 100, where Tb is the torque immedi-
ately before TSI and MVCpeak is the highest MVC value without any
stimulation.
F I G U R E 1 Schematic
overview of the study
JESSEN ET AL. 613

2.7 | SDS page and immunoblotting 2.8 | Statistical analysis

The protein-specific content was determined by Western blotting
as described previously.22 Briefly, freeze-dried muscle tissue
(~2 mg dw) was homogenized for 1 min at 30 Hz on a shaking
bead-mill (TissueLyser II, QIagen, Valencia, CA, USA) in ice-cold
lysis buffer (0.05 mol/L Tris Base pH 7.4, 0.15 mol/L NaCl,
1 mmol/L EDTA and EGTA, 0.05 mol/L sodium fluoride, 5 mmol/L
sodium pyrophosphate, 2 mmol/L sodium orthovanadate, 1 mmol/L
benzamidine, 0.5% protease inhibitor cocktail (P8340, Sigma
Aldrich), and 1% NP-40). The samples were rotated end-over-end
for 30 min at 4 C and centrifuged (18,320 × g) for 20 min at 4 C,
after which the supernatant was collected. The protein concentra-
tion of each sample was determined with a BSA kit (Thermo Fisher
Scientific, MA, US). The samples were mixed with 6 × Laemmli
buffer (7 mL 0.5 M Tris-base, 3 mL glycerol, 0.93 g DTT, 1 g SDS
and 1.2 mg bromophenol blue) and ddH2O to reach an equal pro-
tein concentration. An equal amount of protein was loaded in each
well of pre-casted gels (Bio-Rad Laboratories, CA, US). Samples
from each subject were loaded on the same gel. Bands were visu-
alized with ECL (Millipore, MA, US) and recorded with a digital
camera (ChemiDoc MP Imaging System, Bio-Rad Laboratories, CA,
US). Densitometry quantification of the band intensity was done
using Image Lab version 4.0 (Bio-Rad Laboratories, CA, US) and
determined as the total band intensity adjusted for background
intensity. The primary antibodies used were mTOR (#2972, Cell
Signaling, MA, US), p-mTORSer2448 (#2971, Cell Signaling, MA, US),
and PKA (#9621, Cell Signaling, MA, US). The secondary antibody
used was HRP goat anti-rabbit (Cat. No.: 4010–05, Southern
Biotech, AL, US).
Statistical analyses were performed in SPSS version 26 (IBM software,
Armonk, NY, US). Data are presented as means with standard devia-
tions (SD). To estimate the effect of clenbuterol, a repeated measures
linear mixed maximum likelihood model was used with treatment as
the fixed effect. Outcome statistics are presented as the mean effect
size with 95% confidence intervals and P-values to represent
probability.
3 | RE SU LT S
3.1 | Side effects
Only two subjects reported side effects. One subject reported
tachycardia and muscle tremor, while the other subject reported
tachycardia, dizziness and drowsiness. Heart rate increased by 38%
(P = 0.002) after clenbuterol ingestion, whereas blood pressure and
plasma K+ concentrations did not change significantly (Table 1). No
changes were observed in ECG measurements, apart from elevated
heart rate.
3.2 | Resting metabolic rate
Clenbuterol ingestion increased resting energy expenditure by 21%
(P < 0.001) and fat oxidation by 39% (P = 0.006; Figure 2). Respiratory
exchange ratio (RER) and carbohydrate oxidation did not change sig-
nificantly (P = 0.181 and P = 0.946, respectively; Figure 2).

F I G U R E 2 Individual and
mean values for resting energy
expenditure (A), respiratory
exchange ratio (RER) (B),
carbohydrate oxidation (C), and
fat oxidation (D) before (0 min)
and after (140 min) ingestion of
80 μg clenbuterol. **Different
from 0 min (P < 0.01)
614 JESSEN ET AL.

3.3 | Muscle PKA-signaling and mTORSer2448 3.5 | Skeletal muscle glycogen content
phosphorylation
Glycogen content of the vastus lateralis muscle did not change with
Phosphorylation of mTORSer2448 increased (P = 0.004) by 121%, and clenbuterol ingestion (P = 0.648; Figure 4).
phosphorylation of PKA substrates increased (P = 0.006) by 35% after
clenbuterol ingestion (Figure 3).

3.6 | Plasma concentrations of glucose, lactate,

3.4 | Quadriceps contractile function insulin, and fatty acids

MVC decreased by 4% (P = 0.026) and half-relaxation time shortened Clenbuterol ingestion increased plasma concentrations
by 9% (P = 0.046) after clenbuterol ingestion, whereas voluntary acti- of glucose (+25%; P < 0.001), lactate (+87%; P = 0.004), insu-
vation, time to peak twitch torque, and peak twitch torque did not lin (+105%; P = 0.009), and fatty acids (+129%; P = 0.001)
change significantly after clenbuterol ingestion (Table 2). (Figure 5).

F I G U R E 3 Individual and mean protein content and phosphorylation of mammalian target of rapamycin (mTOR) expressed as ratio of
phosphorylated (p-mTORSer2448) and total protein (A), protein kinase A (PKA) substrate phosphorylation (C), and representative blots (B + D) from
before (0 min) and after (140 min) ingestion of 80 μg clenbuterol. A.u: Arbitrary units. **Different from 0 min (P < 0.01)

TABLE 2 Contractile function of the quadriceps muscle before (0 min) and 140 min after (140 min) ingestion of 80 μg clenbuterol

0 min 140 min Δ-change (95% CI) P-value

MVC (Nm) 277 (± 45) 265 (± 41) −12 (−22 to −2) 0.026
Peak twitch torque (Nm) 94 (± 23) 96 (± 17) 2 (−12 to 16) 0.764
VA (%) 97.8 (± 1.9) 97.8 (± 1.2) 0.1 (−1.1 to 1.0) 0.851
TPT (ms) 65 (± 14) 60 (± 7) −5 (−17 to 7) 0.343
HRT (ms) 47 (± 14) 43 (± 12) −4 (−8 to 0) 0.046

MVC, maximal voluntary contraction torque; VA, voluntary activation; TPT, time to peak twitch; HRT, half-relaxation time. Values are mean (± SD) unless
otherwise specified.
JESSEN ET AL.
F I G U R E 4 Individual and mean values for glycogen content of
the vastus lateralis muscle before (0 min) and after (140 min) ingestion
of 80 μg clenbuterol
4 | DISCUSSION
The novel finding of the present study was that ingestion of 80 μg
clenbuterol exhibited a thermogenic action, increasing fat oxidation,
resulting in a net increase in metabolic rate. Furthermore, the present
study demonstrates that clenbuterol induces mTOR phosphorylation
and PKA-signaling in human skeletal muscle. To our knowledge, this is
the first time the effect of clenbuterol ingestion on resting metabolism
and muscle signaling has been evaluated in humans, despite the fact
that the drug has been misused as a doping agent for many years.
The observed increase in metabolic rate after clenbuterol inges-
tion is higher than that reported after administration of other beta2-
agonists. Onslev et al.17 observed a 12% increase in resting energy
expenditure with inhalation of 54 μg formoterol and a concomitant
38% increase in fat oxidation, and Lee et al. observed increases of
11–13% and 23–25% in resting energy expenditure and fat oxidation,
respectively, after the ingestion of 160 μg oral formoterol. 23,24
F I G U R E 5 Individual and
mean values for plasma glucose
(A), lactate (B), insulin (C), and
fatty acids (D) before (0 min) and
after (140 min) ingestion of 80 μg
clenbuterol. **Different from
0 min (P < 0.01)
615
Likewise, infusion of the short-acting beta2-agonist salbutamol has
been shown to increase energy expenditure by 9–13%25,26 and fat
oxidation by 19%.25 The thermogenic actions of beta2-agonists there-
fore seem attributed to increased fat oxidation. The difference in the
magnitude of the response between beta2-agonists may relate to dif-
ferences in selectivity towards the beta2-receptor, as well as differ-
ences in efficacy and potency.1
The increase in energy expenditure observed with clenbuterol
supports the notion that clenbuterol may be used to reduce fat mass,
as also purported on several doping forums, which are available
through online search engines. In the present study, extrapolation of
the measured energy expenditure indicates that clenbuterol can
increase energy expenditure from approximately 2000 to 2400 kcal/
day. However, care should be taken when extrapolating, as this calcu-
lation assumes that energy expenditure is increased by ~20% for 24 h,
when in fact the action likely diminishes within 24 h after ingestion. It
could reasonably be speculated that the increased metabolic rate after
clenbuterol ingestion lasts longer than that of other beta2-agonists,
such as formoterol or salbutamol, due to the long elimination half-life
of ~25–35 h reported for clenbuterol27,28 compared with ~12 h for
formoterol29 and ~3–4 h for salbutamol.30 Notwithstanding, the ther-
mogenic action of clenbuterol likely translates into a reduction in fat
mass as also observed in rodents31,32 and purported within the body-
building milieu.15
We observed that clenbuterol effectively induced PKA and mTOR
phosphorylation in skeletal muscle. Given that the PKA-dependent
signaling pathway is the canonical signaling pathway associated with
the beta2-adrenoceptor, the observed increase in PKA substrate phos-
phorylation with clenbuterol was expected and was consistent with
that observed for formoterol and salbutamol in human skeletal mus-
cle.33,34 The increase of 121% in mTORSer2448 phosphorylation, how-
ever, was larger than expected, and may indicate anabolic signaling in
616
the absence of exercise. To our knowledge, this is the first study to
demonstrate that a beta2-agonist induces considerable activation of
the mTOR pathway in humans. Beta2-agonist-induced activation of
the mTOR signaling pathway has been shown to be one of the main
mechanisms underlying the apparent ability of these substances to
exert their muscle anabolic actions in rodents.35 It should be noted
that the mTOR pathway may be activated in several ways, such as
protein ingestion or muscle contraction.36 However, our subjects
were fasting until the second muscle biopsy was sampled, and MVC
measurements were only performed for the right leg, while the biopsy
sample was obtained from the left leg, making it unlikely that factors
other than clenbuterol ingestion confounded the phosphorylation
response. Thus, our data indicate that clenbuterol is a potent activator
of mTOR-signaling in human skeletal muscle.
Clenbuterol has been reported to be an effective stimulant for skel-
etal muscle hypertrophy in rodents.10-14 In humans, skeletal muscle
hypertrophy has been reported after chronic treatment with other
beta2-agonists,19,37,38 but only a few studies have investigated the
39
effect of chronic clenbuterol treatment. Maltin et al. reported
increases in muscle strength after 4 weeks of postoperative recovery
with clenbuterol treatment (40 μg/day) compared with a placebo group,
and Koeberl et al.53 reported increases in functional tests after 52 weeks
of clenbuterol treatment (up to 160 μg/day) in patients with Pompe dis-
ease. While neither of these two studies demonstrated clenbuterol-
induced skeletal muscle hypertrophy, the increases in muscle perfor-
mance indicate muscular adaptation to clenbuterol treatment.
40
Kamalakkannan et al. reported increases in lean muscle mass and mus-
cle strength following 12 weeks of clenbuterol treatment (80 μg/day) in
patients with chronic heart failure. Notwithstanding, the side effects
and numerous case reports of cardiac events due to the misuse of
3-7
clenbuterol demonstrate the associated health hazard for any athlete
or amateur using clenbuterol for weight loss or muscle gain.
The glycogen content of the vastus lateralis muscle did not change
with clenbuterol ingestion, which is in agreement with the observed
increases in substrate availability from circulating fatty acids and glu-
cose, as well as an increased concentration of insulin as also shown for
the selective beta2-agonist terbutaline.41 Increases in the plasma con-
centrations of lactate, glucose, fatty acids, and insulin are in agreement
with those observed after administration of other beta2-agonists.41,42
Notably, we observed that MVC declined upon clenbuterol
treatment, which is in contrast to that reported for short-acting
beta2-agonists. For salbutamol and terbutaline, the response seems
to be dose-dependent, with several studies reporting increases in
MVC after high-dose beta2-agonist administration,43-45 while some
studies administering lower doses have reported no effect on
MVC.46,47 The discrepancy between the decrease in MVC observed
for clenbuterol with that observed for short-acting beta2-agonists
may be attributed to beta2-receptor independent mechanisms of
clenbuterol in skeletal muscle. Thus, McCormick et al.48 reported a
decreased force production of isolated mouse muscle fibers per-
fused with clenbuterol, while perfusion with salbutamol increased
force production. In the same study, the beta-receptor antagonist
propranolol reversed the salbutamol-induced increases in force
JESSEN ET AL.
production, but not clenbuterol-induced decreases in force produc-
tion, suggesting beta2-receptor independent impairment of skeletal
muscle force production with clenbuterol.
Because of the relatively large effect size of clenbuterol and low
inter-individual variability, we were able to detect significant effects
of clenbuterol. However, inclusion of a placebo group would have
strengthened the study design. For example, a confounding effect of
fasting on plasma parameters could have been excluded. Neverthe-
less, it seems unlikely that such marked differences in circulating insu-
lin, fatty acids, and glucose should occur within the short span of the
experimental day (~3 h). In fact, the opposite response of what we
observed in the present study would be expected from glucose and
insulin concentrations in response to fasting.49 As the study popula-
tion was a homogenous group of young men, extrapolation to older
individuals may not be possible. Indeed, the response to beta2-ago-
nists may be diminished in older adults50, as also observed in rodent
studies.51,52
In summary, the present findings show that clenbuterol is an effi-
cient thermogenic substance that also induces muscle mTOR phos-
phorylation, indicating that clenbuterol may exert anabolic actions in
human skeletal muscle. Because these effects may translate into a
reduction of fat mass and increased muscle growth, clenbuterol should
continue to be on WADA's list of prohibited substances and methods.
ACKNOWLEDG MENTS
The study was supported by a grant from Anti Doping Denmark. Fig-
ure 1 created with biorender.com.
CONFLIC T OF INT ER E ST
Sara A. Solheim was employed by Anti Doping Denmark during the
intervention and the preparation of this manuscript. Morten Hostrup
and Glenn A. Jacobson have provided independent scientific reports
in antidoping investigations involving beta2-agonists. Both authors are
funded by independent research grants from WADA that pertain to
beta2-agonist pharmacology and physiology.
OR CID
Søren Jessen https://orcid.org/0000-0002-7461-1264
Sara A. Solheim https://orcid.org/0000-0002-4589-1711
Glenn A. Jacobson https://orcid.org/0000-0002-3409-8769
Kasper Eibye https://orcid.org/0000-0002-5737-691X
Jens Bangsbo https://orcid.org/0000-0003-1305-9274
Nikolai B Nordsborg https://orcid.org/0000-0001-7077-9265
Morten Hostrup https://orcid.org/0000-0002-6201-2483
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How to cite this article: Jessen S, Solheim SA, Jacobson GA,
et al. Beta2-adrenergic agonist clenbuterol increases energy
expenditure and fat oxidation, and induces mTOR
phosphorylation in skeletal muscle of young healthy men. Drug
Test Anal. 2020;12:610–618. https://doi.org/10.1002/dta.
2755