Clinical and Translational Report

Exercise-Induced Changes in Visceral Adipose

Tissue Mass Are Regulated by IL-6 Signaling: A
Randomized Controlled Trial
Graphical Abstract Authors
Anne-Sophie Wedell-Neergaard,
Louise Lang Lehrskov,
Regitse Højgaard Christensen, ...,
Bente Klarlund Pedersen,
Helga Ellingsgaard,
Rikke Krogh-Madsen

Correspondence
helga.ellingsgaard@regionh.dk

In Brief
Wedell-Neergaard et al. show that in
abdominally obese people, exercise-
mediated loss of visceral adipose tissue
mass requires IL-6 receptor signaling.
Given that abdominal fat is metabolically
harmful to health, this study raises a
potentially important side effect of IL-6
receptor antibodies, such as tocilizumab,
used to treat some forms of arthritis.

Highlights
d Exercise reduces visceral adipose tissue mass

d Loss of visceral adipose tissue mass following exercise is

dependent on IL-6

d IL-6 receptor blockade increases total cholesterol and is not

influenced by exercise

d Improvements in cardiorespiratory fitness following exercise

are not IL-6 dependent

Wedell-Neergaard et al., 2019, Cell Metabolism 29, 1–12

April 2, 2019 ª 2018 Elsevier Inc.
https://doi.org/10.1016/j.cmet.2018.12.007
 Please cite this article in press as: Wedell-Neergaard et al., Exercise-Induced Changes in Visceral Adipose Tissue Mass Are Regulated by IL-6
Signaling: A Randomized Controlled Trial, Cell Metabolism (2018), https://doi.org/10.1016/j.cmet.2018.12.007

Cell Metabolism

Clinical and Translational Report

Exercise-Induced Changes in Visceral Adipose

Tissue Mass Are Regulated by IL-6 Signaling:
A Randomized Controlled Trial
Anne-Sophie Wedell-Neergaard,1,6 Louise Lang Lehrskov,1,6 Regitse Højgaard Christensen,1,6 Grit Elster Legaard,1
Emma Dorph,1 Monica Korsager Larsen,1 Natja Launbo,1 Sabrina Ravn Fagerlind,1 Sidsel Kofoed Seide,1 Stine Nymand,1
Maria Ball,1 Nicole Vinum,1 Camilla Noerfelt Dahl,1 Marie Henneberg,1 Mathias Ried-Larsen,1 Janus Damm Nybing,2
Robin Christensen,3,4 Jaya Birgitte Rosenmeier,5 Kristian Karstoft,1 Bente Klarlund Pedersen,1 Helga Ellingsgaard,1,7,8,*
and Rikke Krogh-Madsen1,7
1The Centre of Inflammation and Metabolism and the Centre for Physical Activity Research, Rigshospitalet, University of Copenhagen,

2100 Copenhagen, Denmark

2Department of Radiology, Copenhagen University Hospital Bispebjerg, 2400 Copenhagen, Denmark
3Musculoskeletal Statistics Unit, The Parker Institute, Bispebjerg and Frederiksberg Hospital, 2000 Copenhagen, Denmark
4Department of Rheumatology, Odense University Hospital, 5000 Odense, Denmark
5Department of Cardiology, Copenhagen University Hospital Bispebjerg, Copenhagen, 2400 Copenhagen, Denmark
6These authors contributed equally
7These authors contributed equally
8Lead Contact

*Correspondence: helga.ellingsgaard@regionh.dk
https://doi.org/10.1016/j.cmet.2018.12.007

SUMMARY (BMI) (Wedell-Neergaard et al., 2018). Abdominal adiposity re-

Visceral adipose tissue is harmful to metabolic
health. Exercise training reduces visceral adipose
tissue mass, but the underlying mechanisms are
not known. Interleukin-6 (IL-6) stimulates lipolysis
and is released from skeletal muscle during exercise.
We hypothesized that exercise-induced reductions
in visceral adipose tissue mass are mediated by
IL-6. In this randomized placebo-controlled trial, we
assigned abdominally obese adults to tocilizumab
(IL-6 receptor antibody) or placebo during a 12-
week intervention with either bicycle exercise or no
exercise. While exercise reduced visceral adipose
tissue mass, this effect of exercise was abolished in
the presence of IL-6 blockade. Changes in body
weight and total adipose tissue mass showed similar
tendencies, whereas lean body mass did not differ
between groups. Also, IL-6 blockade increased
cholesterol levels, an effect not reversed by exercise.
Thus, IL-6 is required for exercise to reduce visceral
adipose tissue mass and emphasizes a potentially
important metabolic consequence of IL-6 blockade.
INTRODUCTION
Abdominal adiposity is detrimental to health and associated with
an increased risk of cardiometabolic disease (Haffner, 2007),
cancer (Zhang et al., 2008), dementia (Whitmer et al., 2008),
and all-cause mortality (Kuk et al., 2006; Pischon et al., 2008).
Abdominal adiposity is moreover associated with low fitness
and low-grade inflammation independent of body mass index
flects accumulation of visceral adipose tissue, which is located
inside the abdominal cavity and surrounds internal organs like
the liver and the intestines (Després et al., 2001). Visceral adi-
pose tissue is characterized as pro-inflammatory, metabolically
active, and susceptible to lipolysis (Yudkin et al., 2005). While
physical inactivity, without weight gain, leads to accumulation
of visceral adipose tissue (Olsen et al., 2008), physical activity
(exercise training) reduces visceral adipose tissue mass (Nordby
et al., 2012). The mechanisms driving the effect of exercise on
visceral adipose tissue mass remain to be identified.
Interleukin-6 (IL-6) is a cytokine implicated in the regulation of
energy metabolism (Wallenius et al., 2002; Theurich et al., 2017).
IL-6 can be persistently elevated in obese individuals (Cottam
et al., 2004) and acutely following infection (Ma et al., 2016)
and exercise (Ostrowski et al., 1998; Steensberg et al., 2000).
Elevated circulating levels of IL-6 influence both glucose homeo-
stasis (Febbraio et al., 2004; Lang Lehrskov et al., 2018) and lipid
metabolism (Petersen et al., 2005). While the effects of IL-6 on
glucose homeostasis are conflicting (Pedersen and Febbraio,
2007), there is more consensus regarding its role in regulating
lipid metabolism. Infusion of IL-6 into healthy individuals stimu-
lates lipolysis and fatty acid oxidation (van Hall et al., 2003).
In vitro studies in adipocytes and myotubes have confirmed
the effects of IL-6 on lipolysis and fatty acid oxidation, and evi-
dence exists that IL-6 induces these effects by increasing
AMP-activated protein kinase (AMPK) (Carey et al., 2006).
Finally, studies in rodents support a regulatory effect of IL-6 on
adipose tissue mass. Whole-body IL-6 knockout mice develop
mature onset obesity and an overall increase in fat mass, a
phenotype partially reversed by injection of IL-6 (Wallenius
et al., 2002).
Exercise stimulates lipolysis, an effect often attributed to
epinephrine (de Glisezinski et al., 2009). However, given the
lipolytic effect of IL-6, along with the fact that exercise acutely

Cell Metabolism 29, 1–12, April 2, 2019 ª 2018 Elsevier Inc. 1

 Please cite this article in press as: Wedell-Neergaard et al., Exercise-Induced Changes in Visceral Adipose Tissue Mass Are Regulated by IL-6
Signaling: A Randomized Controlled Trial, Cell Metabolism (2018), https://doi.org/10.1016/j.cmet.2018.12.007

Enrolment

Email screening for eligibility (n=592)

Did not meet inclusion criteria (n=264)

Information and informed consent (n=328)

Did not meet inclusion criteria or
failed to provide (n=112)
Phone screening for eligibility (n=216)
Did not meet inclusion criteria (n=128)

Screening: medical examination and blood

samples (n=88)
Did not meet inclusion criteria (n=3)
Other reasons (n=2)
Randomisation (n=83)

Allocation

Allocated to No Exercise Allocated to No exercise Allocated to Exercise + Allocated to Exercise + Allocated to Resistance
+ Placebo (n=18) + Tocilizumab (n=18) Placebo (n=16) Tocilizumab (n=15) training + Placebo (n=16)

Drop-out (n=5) Drop-out (n=3) Drop-out (n=1) Drop-out (n=1) Drop-out (n=2)
Did not adhered to the Did not adhere to the Did not adhere to the Did not adhere to the Did not adhere to the
protocol (n=0) protocol (n=2) protocol (n=1) protocol (n=1) protocol (n=0)

Analyses

Analyzed (n=13) Analyzed (n=13) Analyzed (n=14) Analyzed (n=13)

Figure 1. Flow Chart

increases plasma IL-6 concentrations (Ostrowski et al., 1998;
Steensberg et al., 2000), we hypothesized that exercise medi-
ates its effects on visceral adipose tissue mass through IL-6.
Building upon this hypothesis, we present here a randomized
trial of tocilizumab in abdominally obese adults. Based on a
modified 2 3 2 factorial design, we tested whether combining
exercise training (yes/no) with IL-6 receptor blockade (yes/no)
would hinder exercise-induced reductions in visceral adipose
tissue mass. Abdominally obese men and women performed
12 weeks of endurance exercise or no training with and without
infusions of the IL-6 receptor antagonist tocilizumab. The pri-
mary aim of this specific trial was to evaluate the effect of
12 weeks of endurance exercise and standard dose of tocilizu-
mab on changes in visceral adipose tissue mass.
RESULTS AND DISCUSSION
of exercise modality (strength training versus endurance training)
on cardiac adipose tissue as a secondary and separate
distinctive aim not covered in this paper (Christensen et al.,
2018). Randomization was done using a computer-generated
schedule administered by an independent methodologist to
maintain allocation concealment.
Between August 2016 and April 2018, in total 83 abdominal
obese participants were enrolled in the study and randomly
assigned to one of five groups. However, as described above,
only four groups were included in the prespecified analysis
plan of the primary endpoint—the change in visceral adipose
tissue mass. In these four groups, 14 participants discontinued
and 53 completed the study (Figure 1). As expected from the
study, design baseline characteristics appeared to be similar be-
tween groups; a difference in triglycerides, however, appeared
between groups at baseline (Table 1; see also Table S1).

This was a single-center, randomized, double-blind, parallel Body Composition

group trial (registered on https://clinicaltrials.gov/ as
NCT02901496). Participants were randomized 1:1:1:1:1 in a
modified 2 3 2 factorial design to receive endurance exercise
or no exercise, with or without tocilizumab (IL-6 receptor
blockade); a fifth group performing strength training was
included in the randomization schedule to investigate the effect
Changes in visceral adipose tissue mass were different across
the four groups of interest (p = 0.030). As illustrated in Figure 2,
12 weeks of exercise + placebo induced a decrease in visceral
adipose tissue mass compared to no exercise + placebo
( 225.1g [95% CI: 446.7 to 3.4g]; p = 0.047). However, as
presented in Table 2, when IL-6 signaling was blocked by

2 Cell Metabolism 29, 1–12, April 2, 2019

 Please cite this article in press as: Wedell-Neergaard et al., Exercise-Induced Changes in Visceral Adipose Tissue Mass Are Regulated by IL-6
Signaling: A Randomized Controlled Trial, Cell Metabolism (2018), https://doi.org/10.1016/j.cmet.2018.12.007

Table 1. Baseline Characteristics

No exercise + No exercise + Exercise + Exercise +
placebo tocilizumab placebo tocilizumab Overall
(n = 13) (n = 13) (n = 14) (n = 13) (n = 53)
Female/male (n) 11/2 8 /5 11/3 10/3 40/13
Age (years) 47 (11) 44 (12) 39 (13) 44 (12) 44 (12)
Waist (cm) 106.7 (12.7) 109.9 (14.8) 103.7 (13.8) 106.0 (10.6) 106.6 (12.9)
Waist-to-height ratio 0.63 (0.08) 0.64 (0.08) 0.62 (0.08) 0.62 (0.06) 0.63 (0.07)
BMI (kg2/m) 33.0 (6.3) 32.9 (4.9) 33.0 (4.9) 33.0 (4.7) 33.0 (5.1)
Cardiorespiratory fitness 27 (5) 29 (5) 28 (5) 29 (6) 28 (5)
(mL/min/kg)
Visceral fat mass (g) 2,629 (1,120) 3,518 (2,353) 2,313 (1,423) 2,385 (1,396) 2,705 (1,669)
Total fat mass (g) 43,659 (13,808) 41,944 (9,862) 42,337 (9,529) 41,500 (11,710) 42,335 (10,957)
Android fat mass (g) 4,598 (1,546) 4,463 (1,417) 4,204 (1,308) 4,297 (1,375) 4,383 (1,375)
Gynoid fat mass (g) 7,401 (2,425) 7,047 (1,691) 7,527 (1,718) 7,395 (2,627) 7,345 (2,086)
Lean body mass (g) 47,392 (9,069) 52,696 (11,099) 45,415 (7,369) 49,887 (8,124) 48,810 (91,58)
Body weight (kg) 95.4 (19.9) 98.8 (16.9) 92.4 (13.5) 95.5 (14.5) 95.5 (15.9)
Systemic BP (mmHg) 122 (19) 126 (18) 128 (21) 134 (20) 128 (19)
Diastolic BP (mmHg) 84 (8) 86 (13) 87 (10) 89 (9) 87 (10)
Total cholesterol (mmol/L) 5.06 (0.78) 5.10 (0.98) 4.84 (0.92) 4.81 (0.66) 4.95 (0.83)
LDL cholesterol (mmol/L) 3.28 (0.75) 3.28 (0.85) 3.06 (0.63) 3.08 (0.74) 3.17 (0.73)
Triglyceride (mmol/L) 1.57 (0.58) 1.70 (1.49) 1.16 (0.72) 1.03 (0.33) 1.36 (0.91)
HDL cholesterol (mmol/L) 1.28 (0.35) 1.26 (0.27) 1.39 (0.29) 1.44 (0.26) 1.35 (0.29)
Fasting glucose (mmol/L) 5.0 (0.5) 5.3 (0.9) 5.1 (0.5) 5.0 (0.5) 5.1 (0.6)
Fasting insulin (mmol/L) 100 (40) 92 (58) 111 (78) 88 (55) 98 (59)
Leukocytes (109/L) 6.56 (1.59) 6.44 (1.63) 6.75 (2.20) 6.47 (1.75) 6.56 (1.77)
Neutrophils (109/L) 3.83 (1.33) 3.54 (1.20) 4.01 (1.80) 3.92 (1.46) 3.83 (1.44)
Hs-CRP (mg/L) 3.59 (3.09) 2.01 (1.53) 3.46 (3.51) 2.50 (2.57) 2.89 (2.78)
IL-6 (pg/mL) 0.79 (0.60) 0.98 (0.91) 0.72 (0.26) 0.67 (0.42) 0.79 (0.59)
Smokers (n) 0 2 3 0 5
Alcohol (units/week) 2 (2) 3 (4) 4 (4) 5 (8) 3 (5)
Values are presented as mean (SD) if not noted otherwise. BP, blood pressure; LDL, low-density lipoproteins; HDL, high-density lipoproteins; Hs-CRP,
high-sensitivity C-reactive Protein; IL, interleukin.

tocilizumab, the exercise-induced decrease in visceral adipose
tissue mass abolished. Thus, the exercise + tocilizumab group
increased visceral adipose tissue mass 278.2 g (95% CI 61.69
to 494.73, p = 0.013) more than the exercise + placebo group
(Table 2).
Similar tendencies for changes in total fat mass, android fat
mass, gynoid fat mass, and body weight were observed (Figures
2B–2D and 2F; Table 2). Lean body mass was unchanged in all
four groups following the intervention (Figure 2E; Table 2).
Previous studies have demonstrated reduced visceral adipose
tissue mass following exercise (Sabag et al., 2017). Comparing
the effect size of training-induced changes in visceral adipose
tissue mass in our study with those in the meta-analysis by Sa-
bag et al. (2017) reveals that the effect observed in our study is
small. While we find an 8% reduction in visceral adipose tissue
mass, other studies report 15%–20% reductions. The meta-
analysis indicates that the duration of each exercise bout plays
a role for the magnitude of the effect. The longer the exercise
bout, the greater the reduction. In our study, each exercise ses-
sion lasted 45 min, which is the shortest duration described, and
may possibly explain the small effect size in our study.
Given the lipolytic effect of IL-6 (Petersen et al., 2005), we
hypothesized that exercise-induced IL-6 is mediating the reduc-
tion in visceral adipose tissue mass. Indeed, our study demon-
strates that IL-6 signaling is required for exercise to reduce
visceral adipose tissue mass as the combination of IL-6 receptor
blockade (tocilizumab), preventing IL-6 from signaling, and exer-
cise abolished the effect of exercise on visceral adipose tissue
mass. We can, however, not conclude that IL-6 is driving the
effect of exercise on visceral adipose tissue mass. The present
study also indicates that blocking IL-6 on its own may increase
visceral adipose tissue mass and, hence, implicates IL-6 in the
physiology of visceral adipose tissue regulation. Provided a
continuous increase in visceral adipose tissue mass in the
presence of IL-6 blockade, it may show to be clinically relevant
for patients receiving tocilizumab. Whether tocilizumab inhibits
lipolysis was not assessed in this study.
Given that IL-6 has been implicated in muscle hypertrophy
(Serrano et al., 2008), it is intriguing that IL-6 blockade has no
effect on lean body mass. Thus, the effect of IL-6 blockade
appears to be specific to adipose tissue and, in particular, to
visceral adipose tissue. As visceral adipose tissue has been

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 Please cite this article in press as: Wedell-Neergaard et al., Exercise-Induced Changes in Visceral Adipose Tissue Mass Are Regulated by IL-6
Signaling: A Randomized Controlled Trial, Cell Metabolism (2018), https://doi.org/10.1016/j.cmet.2018.12.007

A ANCOVA: p=0.030 Figure 2. Body Composition

# Changes in (A) visceral fat mass, (B) total fat mass,
300
* (C) android fat mass, (D) gynoid fat mass, (E) lean
Δ Visceral fat mass (g)

no exercise + placebo body mass, and (F) body weight in response to the
200 no exercise + tocilizumab 12-week intervention. Data are presented as least-
square means adjusted for baseline ± SEM (n = 12–
100 exercise + placebo 14 per group). *p = 0.047 comparing no exercise +
placebo versus exercise + placebo. #p = 0.013
0 exercise + tocilizumab comparing exercise + placebo versus exercise +
tocilizumab.
-100
-200
-300 Changes in LDL cholesterol followed
those of total cholesterol (Table 2). There
were differences across the four groups
B ANCOVA: p=0.25 C ANCOVA: p=0.19 D ANCOVA: p=0.93
200 200 (p = 0.032). LDL cholesterol increased in
1000
Δ Android fat mass (g)

Δ Gynoid fat mass (g) the no exercise + tocilizumab group

Δ Total fat mass (g)

500
100 100 (0.29 mM [95% CI 0.07 to 0.52]; p =
0
0.011) as well as in the exercise + tocilizu-
-500 0 0
mab group (0.19 mM [95% CI 0.04 to
-1000 0.41]; p = 0.098) (Table 2). Hence, it was
-100 -100
-1500 not possible to counteract the effect of to-
-2000 -200 -200 cilizumab on LDL and total cholesterol by
exercise (Figure 3B; Table 2). Changes in
HDL cholesterol and triglycerides were
E F
ANCOVA: p=0.56 ANCOVA: p=0.27 not different between groups (Figures 3C
1500 2
Δ Lean body mass (g)

and 3D).
Δ Body weight (kg)

1
Increased total cholesterol and LDL
1000 cholesterol following the tocilizumab inter-
0 vention is in line with previous literature
500 (Smolen et al., 2008) and of a magnitude
-1 that is likely to be of clinical significance
0 -2
(Ference et al., 2017). Exercise had no sig-
nificant effect on lipid profile, and there
were also no clear signs of exercise being
found to express more IL-6 receptors than subcutaneous adi- able to prevent the stimulating effect of IL-6 blockade on total
pose tissue (Ji et al., 2014), it is most likely that visceral adipose cholesterol and LDL cholesterol.
tissue is more sensitive and responsive to changes in IL-6 Fasting blood glucose and plasma insulin also remained
than subcutaneous adipose tissue. However, MRI scans were unchanged following the intervention (Table 2), and there was
used to assess visceral adipose tissue mass, and this approach no significant difference in change between groups (Table 2).
may provide a more accurate measure with less variation Adaptations to exercise training mostly come along with
compared to the DXA scan that was applied to assess total improved insulin sensitivity reflected by decreased plasma insu-
fat mass. Thus, we cannot exclude that the reason for observing lin. Infusion of IL-6 has been reported to lower plasma insulin
differences in visceral fat mass and not total fat mass is (Watt et al., 2005), suggesting that repeated bouts of IL-6,
methodological. induced by exercise, may play a role in mediating enhanced in-
In summary, exercise-induced reductions in visceral adipose sulin sensitivity. Despite no significant changes in plasma insulin
tissue mass are dependent on available IL-6 receptors and, following the intervention, there was a small reduction in insulin in
therefore, on IL-6 signaling. the exercise + placebo group, which, combined with unchanged
glucose, indicates small improvements in insulin sensitivity.
Lipid and Metabolic Profile Interestingly, this effect on insulin was absent in the group that
As illustrated in Figure 3, changes in total cholesterol were combined exercise with tocilizumab; quite in contrast, plasma
different across the four groups (p = 0.014) (Figure 3A). Whereas insulin levels tended to increase in this group, and without
there was no effect on total cholesterol in the no exercise + pla- changes in glucose, these data substantiate a role of IL-6 in
cebo group (0.16 mM [95% CI: 0.15 to 0.46]; p = 0.30) and the training-induced improvements in insulin sensitivity.
exercise + placebo group ( 0.12 mM [95% CI: 0.40 to 0.16];
p = 0.38), an increase in total cholesterol was found in both the Exercise Measures, Cardiorespiratory Fitness
no exercise + tocilizumab group (0.52 mM [95% CI: 0.23 to As illustrated in Figure 4, intervention-induced changes in
0.81]; p = 0.001) and the exercise + tocilizumab group cardiorespiratory fitness (CR-Fitness), maximal oxygen con-
(0.39 mM [95% CI 0.10 to 0.68]; p = 0.10) (Table 2). sumption (VO2max), and VO2max divided by fat-free mass all

4 Cell Metabolism 29, 1–12, April 2, 2019

 Signaling: A Randomized Controlled Trial, Cell Metabolism (2018), https://doi.org/10.1016/j.cmet.2018.12.007
Please cite this article in press as: Wedell-Neergaard et al., Exercise-Induced Changes in Visceral Adipose Tissue Mass Are Regulated by IL-6
Table 2. Summary of Outcome Measures
No exercise + placebo No exercise + tocilizumab Exercise + placebo Exercise + tocilizumab P-ANCOVA Ex + P versus Ex + T p value
Body composition
Visceral fat n = 12 n = 13 n = 14 n = 13 – – –
Week 12(g) 2,788 (2,625 to 2,950) 2,866 (2,706 to 3,027) 2,563 (2,411 to 2,714)a 2,841 (2,684 to 2,997) 0.030 278.2 (61.7 to 494.7) 0.013
Change(g) 82.6 ( 79.7 to 244.9) 161.5 (0.6 to 322.5) 142.4 ( 293.9 to 9.0)a 135.8 ( 20.9 to 292.5) 0.030 278.2 (61.7 to 494.7) 0.013
Relative change (%) 4.9 ( 3.8 to 13.6) 8.6 ( 0.02 to 17.2) 8.0 ( 16.1 to 0.1)a 10.0 (1.6 to 18.4) 0.013 18.0 (6.4 to 29.6) 0.003
Fat mass n = 12 n = 13 n = 14 n = 13 – – –
Week 12 (g) 42,515 (41,421 to 43,609) 42,410 (41,361 41,293 (40,282 42,563 (41,513 0.25 1,269.8 ( 188.0 0.086
to 43,460) to 42,304) to 43,613) to 2,727.5)
Change (g) 180.8 ( 913.3 to 1,274.8) 75.7 ( 973.7 1,041.2 ( 2,052.4 228.5 ( 821.6 0.25 1,269.8 ( 188.0 0.086
to 1,125.2) to 30.1) to 1,278.6) to 2,727.5)
Relative change (%) 0.8 ( 1.9 to 3.5) 0.5 ( 2.1 to 3.1) 2.6 ( 5.1 to 0.1) 0.2 ( 2.8 to 2.4) 0.23 2.4 ( 1.2 to 6.0) 0.18
Android fat mass n = 12 n = 13 n = 14 n = 13 – – –
Week 12 (g) 4,387 (4,260 to 4,514) 4,451 (4,329 to 4,573) 4,267 (4,149 to 4,385) 4,365 (4,243 to 4,487) 0.19 98.4 ( 70.9 to 267.6) 0.25
Change (g) 4.3 ( 122.9 to 131.5) 67.7 ( 54.2 to 189.6) 116.1 ( 233.9 to 1.6) 17.8 ( 139.7 to 104.1) 0.19 98.4 ( 70.9 to 267.6) 0.25
Relative change (%) 1.0 ( 2.2 to 4.1) 1.9 ( 1.2 to 4.8) 3.0 ( 5.9 to 0.1) 0.9 ( 3.9 to 2.1) 0.10 2.1 ( 2.0 to 6.3) 0.31
Gynoid fat mass n = 12 n = 13 n = 14 n = 13 – – –
Week 12 (g) 7,310 (7,081 to 7,538) 7,367 (7,147 to 7,587) 7,272 (7,060 to 7,484) 7,336 (7,117 to 7,556) 0.93 64.5 ( 240.6 to 369.7) 0.67
Change (g) 35.4 ( 264.1 to 193.3) 22.0 ( 198.3 to 242.2) 73.3 ( 285.2 to 138.6) 8.8 ( 228.5 to 210.9) 0.93 64.5 ( 240.6 to 369.7) 0.67
Relative change (%) 0.4 ( 3.5 to 2.8) 0.8 ( 2.3 to 3.8) 1.3 ( 4.3 to 1.6) 1.0 ( 4.1 to 2.0) 0.77 0.3 ( 3.9 to 4.6) 0.88
Lean body mass n = 12 n = 13 n = 14 n = 13 – – –
Week 12 (g) 49,399 (48,620 to 50,177) 49,107 (48,343 49,848 (49,115 49,594 (48,847 0.56 253.6 ( 1306.5 0.63
to 49,871) to 50,581) to 50,341) to 799.2)
Change (g) 589.1 ( 189.0 to 1,367.3) 297.5 ( 466.5 1,038.3 (305.3 784.6 (37.9 to 1,531.4) 0.56 253.6 ( 1306.5 0.63
to 1,061.4) to 1,771.2) to 799.2)
Cell Metabolism 29, 1–12, April 2, 2019 5

Relative change (%) 1.1 ( 0.5 to 2.8) 0.9 ( 0.8 to 2.5) 2.3 (0.7 to 3.8) 1.8 (0.2 to 3.4) 0.62 0.5 ( 2.7 to 1.8) 0.67
Body weight n = 12 n = 13 n = 14 n = 13 – – –
Week 12 (kg) 96.1 (94.9 to 97.3) 96.2 (95.0 to 97.4) 94.9 (93.7 to 96.0) 96.4 (95.2 to 97.6) 0.27 1.5 ( 0.2 to 3.2) 0.079
Change (kg) 0.6 ( 0.6 to 1.9) 0.7 ( 0.5 to 1.9) 0.6 ( 1.7 to 0.6) 0.9 ( 0.3 to 2.1) 0.27 1.5 ( 0.2 to 3.2) 0.079
Relative change (%) 0.8 ( 0.6 to 2.1) 0.8 ( 0.5 to 2.1) 0.7 ( 1.9 to 0.6) 0.9 ( 0.4 to 2.2) 0.27 1.5 ( 0.3 to 3.3) 0.091
Lipid and metabolic profile
Total cholesterol n = 12 n = 13 n = 14 n = 13 – – –
Week 12 (mmol/L) 5.10 (4.80 to 5.41) 5.47 (5.18 to 5.76) 4.82 (4.54 to 5.10) 5.33 (5.04 to 5.63) 0.014 0.51 (0.11 to 0.92) 0.014
Change (mmol/L) 0.16 ( 0.15 to 0.46) 0.52 (0.23 to 0.81) 0.12 ( 0.40 to 0.16) 0.39 (0.10 to 0.68) 0.014 0.51 (0.11 to 0.92) 0.014
(Continued on next page)
6 Cell Metabolism 29, 1–12, April 2, 2019

Table 2. Continued

Signaling: A Randomized Controlled Trial, Cell Metabolism (2018), https://doi.org/10.1016/j.cmet.2018.12.007

Please cite this article in press as: Wedell-Neergaard et al., Exercise-Induced Changes in Visceral Adipose Tissue Mass Are Regulated by IL-6
No exercise + placebo No exercise + tocilizumab Exercise + placebo Exercise + tocilizumab P-ANCOVA Ex + P versus Ex + T p value
Relative change (%) 3.07 ( 3.19 to 9.32) 10.92 (4.89 to 16.94) 2.03 ( 7.83 to 3.76) 9.14 (3.12 to 15.15) 0.012 11.17 (2.84 to 19.50) 0.010
LDL cholesterol n = 12 n = 13 n = 14 n = 13 – – –
Week 12 (mmol/L) 3.30 (3.07 to 3.53) 3.46 (3.24 to 3.69) 3.01 (2.79 to 3.22) 3.36 (3.13 to 3.58) 0.032 0.35 (0.04 to 0.66) 0.029
Change (mmol/L) 0.13 ( 0.10 to 0.36) 0.29 (0.07 to 0.52) 0.16 ( 0.38 to 0.06) 0.19 ( 0.04 to 0.41) 0.032 0.35 (0.04 to 0.66) 0.029
Relative change (%) 5.22 ( 2.81 to 13.25) 10.70 (2.98 to 18.43) 5.28 ( 12.72 to 2.17) 9.46 (1.75 to 17.18) 0.017 14.74 (4.05 to 25.43 0.008
Triglycerides n = 12 n = 13 n = 14 n = 13 – ––
Week 12 (mmol/L) 1.41 (1.01 to 1.81) 1.77 (1.38 to 2.15) 1.37 (1.00 to 1.75) 1.89 (1.50 to 2.28) 0.16 0.51 ( 0.02 to 1.04) 0.058
Change (mmol/L) 0.06 ( 0.34 to 0.46) 0.41 (0.02 to 0.80) 0.02 ( 0.35 to 0.39) 0.53 (0.14 to 0.92) 0.16 0.51 ( 0.02 to 1.04) 0.058
Relative change (%) 13.56 ( 15.54 to 42.66) 24.24 ( 4.08 to 52.55) 6.26 ( 20.68 to 33.19) 47.94 (19.67 to 76.21) 0.17 41.69 (3.07 to 80.31) 0.035
HDL cholesterol n = 12 n = 13 n = 14 n = 13 – – –
Week 12 (mmol/L) 1.36 (1.27 to 1.45) 1.40 (1.31 to 1.48) 1.32 (1.24 to 1.40) 1.39 (1.30 to 1.47) 0.55 0.07 ( 0.05 to 0.18) 0.25
Change (mmol/L) 0.01 ( 0.07 to 0.10) 0.05 ( 0.03 to 0.13) 0.03 ( 0.10 to 0.05) 0.04 ( 0.04 to 0.13) 0.55 0.07 ( 0.05 to 0.18) 0.25
Relative change (%) 1.76 ( 4.28 to 7.79) 2.93 ( 2.89 to 8.75) 1.07 ( 6.64 to 4.50) 2.47 ( 3.37 to 8.31) 0.75 3.54 ( 4.47 to 11.55) 0.38
Fasting blood glucose n = 12 n = 13 n = 14 n = 13
Week 12 (mmol/L) 5.14 (4.91 to 5.38) 5.25 (5.03 to 5.48) 5.18 (4.96 to 5.39) 5.03 (4.81 to 5.25) 0.56 0.15 ( 0.46 to 0.16) 0.34
Change (mmol/L) 0.06 ( 0.17 to 0.30) 0.17 ( 0.05 to 0.4) 0.10 ( 0.12 to 0.31) 0.05 ( 0.27 to 0.17) 0.56 0.15 ( 0.46 to 0.16) 0.34
Relative change (%) 1.43 ( 3.27 to 6.14) 3.69 ( 0.87 to 8.25) 1.75 ( 2.58 to 6.10) 0.28 ( 4.80 to 4.24) 0.67 2.04 ( 8.31 to 4.22) 0.52
Fasting Insulin n = 12 n = 13 n = 14 n = 13 – – –
Week 12 (mmol/L) 114.2 (88.3 to 140.1) 100.2 (75.3 to 125.1) 84.9 (60.8 to 109.1) 99.5 (74.5 to 124.5) 0.44 14.5 ( 20.3 to 49.4) 0.41
Change (mmol/L) 16.4 ( 9.4 to 42.4) 2.5 ( 22.3 to 27.4) 12.7 ( 36.9 to 11.3) 1.8 ( 23.1 to 26.7) 0.44 14.5 ( 20.3 to 49.4) 0.41
Relative change (%) 2.4 ( 21.9 to 26.8) 14.8 ( 8.5 to 38.3) 6.3 ( 29.0 to 16.4) 14.9 ( 8.5 to 38.5) 0.50 21.3 ( 11.5 to 54.2) 0.20
Systolic BP n = 12 n = 13 n = 14 n = 13 – – –
Week 12 (mmHg) 125.8 (119.1 to 132.6) 129.0 (122.6 to 135.5) 127.2 (121.0 to 133.5) 130.1 (123.0 to 137.3) 0.82 2.8 ( 6.5 to 12.3) 0.54
Change (mmHg) 2.2 ( 9.0 to 4.5) 0.9 ( 5.4 to 7.4) 0.8 ( 7.0 to 5.4) 2.0 ( 5.0 to 9.2) 0.82 2.8 ( 6.5 to 12.3) 0.54
Relative change (%) 0.1 ( 5.3 to 5.5) 1.1 ( 4.0 to 6.3) 1.3 ( 3.6 to 6.3) 2.1 ( 3.5 to 7.9) 0.96 0.8 ( 6.8 to 8.4) 0.83
Diastolic BP n = 12 n = 13 n = 14 n = 13 – – –
Week 12 (mmHg) 86.6 (80.9 to 92.3) 82.4 (77.0 to 87.8) 83.0 (77.8 to 88.2) 83.4 (77.5 to 89.4) 0.72 0.4 ( 7.4 to 8.3) 0.92
Change (mmHg) 0.1 ( 5.7 to 5.6) 4.2 ( 9.6 to 1.1) 3.6 ( 8.8 to 1.5) 3.2 ( 9.1 to 2.7) 0.72 0.4 ( 7.4 to 8.3) 0.92
Relative change (%) 0.8 ( 5.4 to 7.1) 4.2 ( 10.3 to 1.7) 3.9 ( 9.7 to 1.9) 3.2 ( 9.9 to 3.3) 0.63 0.6 ( 8.1 to 9.4) 0.89
Exercise measures
Vo2max n = 11 n = 12 n = 14 n = 13 – – –
Week 12 (mL/min) 2,597.30 (2,448.60 2,495.69 (2,352.08 2872.39 (2741.65 2923.60 (2788.51 <0.001 51.22 ( 137.51 0.59
to 2,746.00) to 2,639.29) to 3003.12)a to 3058.70)b to 239.94)
Change (mL/min) 89.13 ( 237.83 to 59.57) 190.74 ( 334.35 185.96 (55.22 237.17 (102.08 <0.001 51.22 ( 137.51 0.59
to 47.13) to 316.69)a to 372.27)b to 239.94)
Relative change (%) 3.06 ( 8.13 to 2.02) 5.91 ( 10.82 to 1.01) 7.51 (3.04 to 11.97)a 10.66 (6.05 to 15.27)b <0.001 3.15 ( 3.29 to 9.60) 0.33
(Continued on next page)
 Please cite this article in press as: Wedell-Neergaard et al., Exercise-Induced Changes in Visceral Adipose Tissue Mass Are Regulated by IL-6
Signaling: A Randomized Controlled Trial, Cell Metabolism (2018), https://doi.org/10.1016/j.cmet.2018.12.007

Values are presented as least-square means (means adjusted for baseline) with 95% CI by treatment group. ANCOVA, analysis of covariance; P, placebo; T, tocilizumab; Ex, exercise; Week 12,
differed between groups (Table 2). Cardiorespiratory fitness
increased 4.44 mL/min/kg (95% CI: 2.15 to 6.74; p < 0.001) in
p value

0.80
0.80
0.92

0.63
0.63
0.53

0.82

0.82
0.95
the exercise + placebo group and 3.11 mL/min/kg (95% CI:
–

–
0.80 to 5.42; p = 0.009) in the exercise + tocilizumab group. Im-
provements in cardiorespiratory fitness did not differ between

1.35 ( 10.50 to 13.21)

0.28 ( 2.49 to 1.93)
0.28 ( 2.49 to 1.93)
Ex + P versus Ex + T

0.40 ( 7.15 to 7.95)

0.97 ( 3.05 to 4.98)

0.97 ( 3.05 to 4.98)
2.31 ( 4.99 to 9.61)

0.22 ( 6.43 to 6.86)

these two groups (Table 2). Changes in VO2max and maximum
oxygen uptake divided by the fat-free mass showed comparable

1.352 ( 10.50
to CR-Fitness (Table 2).
All participants in the two training groups complied with the
to 13.21) minimum of 29 training sessions; the exercise + placebo group
performed 32 (2) sessions and the exercise + tocilizumab group
–

performed 33 (3) sessions. During exercise, the average heart

P-ANCOVA

rate (146 beats/min [SD: 12]) and the maximal heart rate
posttest; LDL, low-density lipoproteins; HDL, high-density lipoproteins; BP, blood pressure; VO2max, maximal oxygen consumption; FFM, fat-free mass.
(168 beats/min [SD: 11]) were similar in the exercise + placebo
<0.001
<0.001
<0.001

<0.001
<0.001
<0.001

<0.001

<0.001
<0.001

group and the exercise + tocilizumab group. This was in line

with the reported no difference in the perceived exertion during
–

an exercise bout (Borg Scale, 16 [SD: 2]).

30.79 (29.20 to 32.38)b

59.55 (56.66 to 62.44)b

25.77 (17.26 to 34.27)b

Exercise + tocilizumab

Studies in IL-6 knockout mice have shown reduced exercise

13.43 (8.66 to 18.19)b
9.42 (3.99 to 14.84)b

8.26 (3.01 to 13.51)b

€ldt et al., 2004); however,

b

4.22 (1.33 to 7.11)b

endurance in the absence of IL-6 (Fa

2.22 (0.63 to 3.81)

in this human study, lacking the IL-6 signal did not seem to influ-
223.57 (215.06

ence the objective and subjective parameters related to exercise

to 232.07)b

performance. Moreover, the improvements in cardiorespiratory

n = 13

n = 13

n = 13

fitness were comparable between the two exercising groups,

as were the total amount of exercise and the intensity of each ex-
ercise bout (see Table S2).
Plasma IL-6 levels increased in response to a single bout of
31.07 (29.54 to 32.60)a

58.58 (55.79 to 61.37)a

24.41 (16.28 to 32.55)a

a
13.21 (8.65 to 17.77)
9.02 (3.79 to 14.25)a

5.95 (0.88 to 11.02)a

exercise (see Table S2). Also, epinephrine and norepinephrine

Exercise + placebo

a
2.50 (0.96 to 4.03)

3.25 (0.46 to 6.04)

increased during the exercise bout (see Table S2). After control-
222.21 (214.08

ling for baseline values, there was no difference in exercise-

to 230.35)a

induced increases of plasma epinephrine (60.6 [95% CI: 20.9

to 142.1]; p = 0.14) or plasma norepinephrine (121.2 [95%
n = 14

n = 14

n = 14

CI: 304.1 to 546.5]; p = 0.6) between the two exercising groups.

Finally, to ensure that the observed changes were not due to
No exercise + tocilizumab

compensatory changes in free-living physical activity and dietary

2.11)

2.86)
0.57)

1.37)

4.75 ( 13.54 to 4.03)

intake, these were monitored before, during, and after the inter-
26.34 (24.68 to 28.01)

50.95 (47.95 to 53.96)

2.30 ( 7.23 to 2.62)

vention. There were no significant differences in free-living daily

193.05 (184.2646
7.80 ( 13.49 to

8.33 ( 13.79 to
2.23 ( 3.90 to

4.38 ( 7.38 to

physical activity before and after the intervention; however, dur-

ing the intervention, both groups treated with tocilizumab
to 201.83)

seemed to spend more time in the moderate and vigorous

n = 12

n = 12

n = 12

physical activity category than the placebo-treated groups.

There were no differences in dietary intake between the groups
(see Table S3).
Exercise-induced reductions in visceral adipose tissue have
p < 0.05 in comparison to no exercise + tocilizumab.
No exercise + placebo

been suggested to be mediated by epinephrine (Enevoldsen

27.96 (26.23 to 29.70)

54.01 (50.85 to 57.17)

0.61 ( 2.35 to 1.12)
2.80 ( 8.73 to 3.13)

1.32 ( 4.48 to 1.84)

2.65 ( 8.39 to 3.09)

p < 0.05 in comparison to no exercise + placebo.

0.93 ( 8.32 to 10.18)
0.85 ( 4.33 to 6.04)

et al., 2000). We did not find a difference in the systemic concen-

tration of exercise-induced epinephrine in the two exercising
198.73 (189.48

groups; whether there were local differences has not been as-
to 207.98)

sessed. Hence, in this study, systemic epinephrine on its own

n = 11

n = 11

n = 11

probably did not play a major role in mediating the observed

effects on visceral adipose tissue.
Week 12 (mL/min/FFM)

Leukocytes and Cytokines

Change (mL/min/FFM)
Cardiorespiratory fitness
Week 12 (mL/min/kg)
Continued

Change (mL/min/kg)
Relative change (%)

Relative change (%)

Relative change (%)

Table 3 presents reduced leucocyte and neutrophil granulocyte

(neutrophils) counts in response to tocilizumab. This effect
tocilizumab was observed both with and without exercise, and
Week 12 (W)
VO2max/ FFM

Change (W)

there was no difference in change between these two groups.

Watt max
Table 2.

Tocilizumab increased plasma concentrations of IL-6 and the

soluble IL-6 receptor (Table 3). These effects of tocilizumab
on both neutrophils and IL-6 are compatible with previous
b
a

Cell Metabolism 29, 1–12, April 2, 2019 7

 Please cite this article in press as: Wedell-Neergaard et al., Exercise-Induced Changes in Visceral Adipose Tissue Mass Are Regulated by IL-6
Signaling: A Randomized Controlled Trial, Cell Metabolism (2018), https://doi.org/10.1016/j.cmet.2018.12.007

A B Figure 3. Lipid Profile

ANCOVA: p=0.014 ANCOVA: p=0.032 Changes in (A) total cholesterol, (B) LDL cholesterol,
Δ Total Cholesterol (mmol/L)

Δ LDL Cholesterol (mmol/L)

0.8 * 0.6 * (C) HDL cholesterol, and (D) triglycerides in
no exercise + placebo
0.6 0.4
response to the 12-week intervention. Data are
no exercise + tocilizumab
presented as least-square means adjusted for
0.4 exercise + placebo
0.2 baseline ± SEM (n = 12–14 per group). *p = 0.029
0.2 exercise + tocilizumab comparing exercise + placebo versus exercise +
0.0
0.0 tocilizumab. #p = 0.014 comparing exercise + pla-
-0.2 -0.2 cebo versus exercise + tocilizumab.

-0.4 -0.4

C ANCOVA: p=0.55
D ANCOVA: p=0.16
the overall changes in IL-1Ra and IL-10 were
Δ HDL Cholesterol (mmol/L)

0.10 0.8 not different between groups (Table 3).

Δ Triglycerides (mmol/L)

Comparing changes in circulating concen-

0.05
0.00
-0.05
-0.10
reports (Suwa et al., 2000; Lang Lehrskov et al., 2018; Moots
et al., 2017).
Neither leucocytes and neutrophils nor IL-6 and the soluble
IL-6 receptor changed in response to exercise + placebo (Ta-
ble 3). Similarly, changes in high-sensitivity C-reactive protein
(hsCRP) were related to tocilizumab rather than exercise. Given
that IL-6 is the main inducer of CRP (Unver and McAllister, 2018),
it was to be expected that tocilizumab reduced hsCRP. The fact
that exercise had no effect on baseline hsCRP or IL-6 (Table 3) is
in accordance with some studies showing no effect of exercise
training while other studies find that baseline hsCRP and IL-6
decrease with exercise training (Benatti and Pedersen, 2015;
Petersen and Pedersen, 2005).
IL-6 has been shown to enhance systemic concentrations of
IL-1Ra and IL-10 (Steensberg et al., 2003); however, in this study,
0.6 trations of adiponectin, leptin, IL-18, IFN-g,
IL-1b, IL-8, and TNF-a between groups
0.4
showed no differences (see Table S4).
0.2
Conclusion and Perspectives
0.0 In addition to exercise-induced IL-6,
chronic low-grade elevations of IL-6 are
seen in patients with severe obesity, type 2 diabetes, and cardio-
vascular disease (Fox et al., 2007). Under these conditions,
elevated IL-6 likely reflects an ongoing TNF-alpha replication
(Benatti and Pedersen, 2015). The signaling pathways, both up-
stream and downstream, in immune cells versus muscle cells
differ markedly and result in pro-inflammatory versus anti-in-
flammatory actions (Pedersen and Febbraio, 2008). It is therefore
likely that IL-6 may act differently in healthy and diseased people.
Nevertheless, this rigorously designed experimental trial
enabled causal inference; we show by blocking the IL-6 receptor
that IL-6 signaling is required for exercise to reduce visceral
adipose tissue mass in abdominal obese humans. The study
moreover demonstrated that blocking IL-6 signaling in already
abdominally obese humans reduced hsCRP and increased
visceral adipose tissue mass, as well as total cholesterol and

A ANCOVA: p<0.001 B ANCOVA: p<0.001 Figure 4. Cardiorespiratory Fitness

# Changes in (A) cardiorespiratory fitness (CR-
* #
Δ CR-Fitness (mL/min/kg)

4 400
* no exercise + placebo fitness), (B) maximal oxygen consumption
Δ VO2max (mL/min)

(VO2max), (C) VO2max divided by fat-free mass

2 200 no exercise + tocilizumab
(FFM), and (D) watt max. Data were presented as
exercise + placebo
means adjusted for baseline (least-square means) ±
0 0 exercise + tocilizumab
SEM (n = 11–14 per group). * indicates a significant
difference between no exercise + placebo versus
-2 -200
exercise + placebo (p < 0.05). # indicates a signifi-
cant difference between no exercise + tocilizumab
-4 -400
versus exercise + tocilizumab (p < 0.001). See also
Table S2.
C D
ANCOVA: p<0.001 ANCOVA: p<0.001
Δ VO2max/ FFM (mL/min/kg)

10 # 40 #
* *
30
Δ Watt max (W)

5
20
0
10
-5
0

-10 -10

8 Cell Metabolism 29, 1–12, April 2, 2019

 Signaling: A Randomized Controlled Trial, Cell Metabolism (2018), https://doi.org/10.1016/j.cmet.2018.12.007
Please cite this article in press as: Wedell-Neergaard et al., Exercise-Induced Changes in Visceral Adipose Tissue Mass Are Regulated by IL-6
Table 3. Change in Leukocytes and Cytokines
No exercise + placebo No exercise + tocilizumab Exercise + placebo Exercise + tocilizumab P-ANCOVA Ex + P versus Ex + T p value
Leucocytes n = 12 n = 13 n = 14 n = 13 – – –
Week 12 (109/L) 6.66 (6.00 to 7.32) 4.91 (4.27 to 5.54) 6.75 (6.14 to 7.37) 5.21 (4.58 to 5.85) <0.001 1.54 ( 2.43 to 0.66) 0.001
Change (109/L) 0.10 ( 0.56 to 0.76) 1.65 ( 2.29 to 1.01) 0.20 ( 0.42 to 0.81) 1.35 ( 1.98 to 0.71) <0.001 1.54 ( 2.43 to 0.66) 0.001
Relative change (%) 4.67 ( 5.52 to 14.85) 23.42 ( 33.21 to 13.63) 3.97 ( 5.48 to 13.41) 19.83 ( 29.62 to 10.04) <0.001 23.80 ( 37.41 to 10.18) 0.001
Neutrophils n = 12 n = 13 n = 14 n = 13 – – –
Week 12 3.88 (3.39 to 4.38) 2.30 (1.82 to 2.78) 3.79 (3.33 to 4.25) 2.49 (2.02 to 2.97) <0.001 1.30 ( 1.96 to 0.64) <0.001
Change (109/L) 0.06 ( 0.44 to 0.55) 1.53 ( 2.01 to 1.05) 0.03 ( 0.49 to 0.43) 1.33 ( 1.81 to 0.86) <0.001 1.30 ( 1.96 to 0.64) <0.001
Relative change (%) 7.54 ( 5.88 to 20.97) 38.65 ( 51.61 to 25.69) 1.78 ( 10.67 to 14.23) 32.06 ( 44.96 to 19.16) <0.001 33.84 ( 51.75 to 15.93) <0.001
Hs-CRP n = 12 n = 13 n = 14 n = 13 – – –
Week 12 3.05 (1.89 to 4.21) 1.05 ( 0.07 to 2.17) 3.03 (1.96 to 4.10) 0.83 ( 0.28 to 1.94) 0.0063 2.21 ( 3.75 to 0.66) 0.006
Change (mg/L) 0.16 ( 1.00 to 1.32) 1.83 ( 2.95 to 0.71) 0.14 ( 0.93 to 1.22) 2.06 ( 3.17 to 0.95) 0.0063 2.21 ( 3.75 to 0.66) 0.006
Relative change (%) 10.23 ( 21.84 to 42.30) 67.91 ( 98.88 to 36.94) 5.79 ( 23.85 to 35.42) 67.32 ( 97.97 to 36.67) <0.001 73.10 ( 115.91 0.001
to 30.30)
IL-6 n = 12 n = 13 n = 14 n = 13 – – –
Week 12 0.74 ( 0.87 to 2.36) 8.59 (7.15 to 10.02) 0.95 ( 0.42 to 2.32) 9.35 (7.93 to 10.78) <0.001 8.40 (6.44 to 10.37) <0.001
Change (pg/mL) 0.05 ( 1.67 to 1.56) 7.79 (6.36 to 9.23) 0.15 ( 1.21 to 1.52) 8.56 (7.14 to 9.98) <0.001 8.41 (6.44 to 10.37) <0.001
Relative change (%) 167.75 ( 53.75 1,368.31 (1,161.63 31.51 ( 228.24 1,288.04 (1,083.19 <0.001 1,319.55 (1,036.45 <0.001
to 389.25) to 1,575.00) to 165.22) to 1,492.89) to 1,602.65)
Soluble IL-6 receptor n = 12 n = 13 n = 14 n = 13 – – –
Week 12 28.57 (22.70 to 34.45) 122.91 (117.46 to 128.36) 29.22 (23.95 to 34.50) 132.77 (127.31 to 138.24) <0.001 103.55 (95.99 to 111.10) <0.001
Change (ng/mL) 1.09 ( 6.97 to 4.78) 93.24 (87.79 to 98.69) 0.44 ( 5.71 to 4.83) 103.11 (97.64 to 108.57) <0.001 103.55 (95.99 to 111.10) <0.001
Relative change (%) 10.93 ( 21.18 to 43.05) 325.53 (295.75 to 355.31) 6.74 ( 35.56 to 22.07) 379.02 (349.17 to 408.87) <0.001 385.77 (344.48 to 427.05) <0.001
IL-1Ra n = 12 n = 13 n = 14 n = 13 – – –
Week 12 742.77 (461.34 353.14 (82.82 to 623.46) 343.58 (81.54 to 605.63)a 360.86 (88.93 to 632.79) 0.13 17.28 ( 362.81 to 397.37) 0.93
to 1,024.19)
Cell Metabolism 29, 1–12, April 2, 2019 9

Change (pg/L) 319.2376 (37.81 70.39 ( 340.71 79.95 ( 341.99 62.67 ( 334.60 0.13 17.28 ( 362.81 to 397.37) 0.93
to 600.66) to 199.93) to 182.10)a to 209.26)
Relative change (%) 63.28 (25.22 to 101.33) 5.69 ( 42.24 to 30.87) 1.42 ( 34.01 to 36.86)a 2.66 ( 39.43 to 34.12) 0.036 4.08 ( 55.48 to 47.32) 0.874
IL-10 n = 12 n = 13 n = 14 n = 13 – – –
Week 12 0.39 (0.27 to 0.51) 0.23 (0.11 to 0.35) 0.23 (0.12 to 0.34)a 0.28 (0.16 to 0.39) 0.18 0.05 ( 0.11 to 0.21) 0.52
Change (pg/L) 0.17 (0.06 to 0.30) 0.02 ( 0.10 to 0.13) 0.01 ( 0.10 to 0.12)a 0.06 ( 0.05 to 0.18) 0.18 0.05 ( 0.11 to 0.21) 0.52
Relative change (%) 90.76 (30.97 to 150.55) 22.48 ( 35.03 to 79.99) 13.82 ( 41.23 to 68.88) 26.66 ( 30.49 to 83.82) 0.24 12.84 ( 66.55 to 92.23) 0.75
Values are presented as least-square means (means adjusted for baseline) with 95% CI by treatment group. ANCOVA, analysis of covariance; P, placebo; T, tocilizumab; Ex, exercise; Week 12,
posttest; Hs-CRP, high-sensitivity C-reactive protein; IL, interleukin; Ra, receptor antagonist.
a
p < 0.05 in comparison to no exercise + placebo.
 Please cite this article in press as: Wedell-Neergaard et al., Exercise-Induced Changes in Visceral Adipose Tissue Mass Are Regulated by IL-6
Signaling: A Randomized Controlled Trial, Cell Metabolism (2018), https://doi.org/10.1016/j.cmet.2018.12.007
LDL cholesterol, independently of exercise. Hence, this study
has consolidated a physiological role of IL-6 as a lipolytic factor
in humans with abdominal obesity and highlights a potentially
important metabolic consequence of IL-6 blockade.
Adverse Events
Overall, 47 adverse events in 36 participants occurred. None of
them were serious adverse events (as defined by the Interna-
tional Conference on Harmonization of Technical Requirements
for Registration of Pharmaceuticals for Human Use, Current Step
2 version 11th June 2015). There were no more registered events
of headache, viral infections, or antibiotic-requiring infections in
the tocilizumab-treated groups than in the placebo-treated
groups (see Table S5).
Limitations of the Study
The potential bias introduced by the per protocol analysis may be
viewed as a limitation. However, since the study is investigating a
mechanism of action, this analysis is justified. Also, our study is
an explorative study not intended to evaluate a given treatment.
The rather low number of participants may be viewed as a limita-
tion; yet, given that the results are clear despite a rather hetero-
geneous population (sex, age), this may be viewed as a strength.
The fact that we do not consider the sex-dependent fat distribu-
tion in our analysis may moreover be viewed as a limitation. Study
participants were all abdominally obese; accordingly, it remains
to be clarified whether IL-6 regulates visceral adipose tissue
mass in humans with all waist circumferences.
STAR+METHODS
Detailed methods are provided in the online version of this paper
and include the following:
d KEY RESOURCES TABLE
d CONTACT FOR REAGENT AND RESOURCE SHARING
d EXPERIMENTAL MODEL AND PARTICIPANT DETAILS
B Study Design, Participants, and Setting
B Sequence Generation and Allocation Concealment
B Interventions
d METHOD DETAILS
B Anthropometrics and Blood Pressure Measurements
B Body Composition and Analysis
B Physical Fitness
B Free-living Physical Activity and Diet
B Tocilizumab and Placebo Infusions
B Blood Samples
d QUANTIFICATION AND STATISTICAL ANALYSIS
B Sample Size and Power Considerations
B Statistical Analysis
SUPPLEMENTAL INFORMATION
Supplemental Information includes five tables and can be found with this
article online at https://doi.org/10.1016/j.cmet.2018.12.007.
ACKNOWLEDGMENTS
This study was funded by TrygFonden. The Centre for Physical Activity
Research (CFAS) is supported by a grant from TrygFonden, and the Centre
10 Cell Metabolism 29, 1–12, April 2, 2019
of Inflammation and Metabolism (CIM) is a member of DD2 – the Danish Center
for Strategic Research in Type 2 Diabetes (the Danish Council for Strategic
Research, grant no. 09-067009 and 09-075724). Musculoskeletal Statistics
Unit, the Parker Institute, Bispebjerg and Frederiksberg Hospital (RC) is sup-
ported by a core grant from the Oak Foundation (OCAY-13-309). L.L.L. was
partly financed by a grant from the Danish Diabetes Academy, which is sup-
ported by the Novo Nordisk Foundation. R.H.C. was supported by the Danish
Heart Foundation (grant number 16-R107-A6704-22970).
AUTHOR CONTRIBUTIONS
Conceptualization, H.E., R.K.-M., B.K.P., J.B.R., and K.K.; Methodology,
A.-S.W.-N., L.L.L., R.H.C., J.D.N., M.R.-L., K.K., H.E., R.K.-M., and B.K.P.;
Formal Analysis, A.-S.W.-N., R.C., and R.K.-M.; Investigation, A.-S.W.-N.,
L.L.L., R.H.C., G.E.L., M.H., E.D., M.K.L., N.L., M.R.-L., S.R.F., S.K.S., S.N.,
M.B., N.V., and C.N.D.; Resources, B.K.P.; Writing – Original Draft,
A.-S.W.-N., H.E., B.K.P., and R.K.-M.; Writing – Review & Editing, all authors
contributed; Visualization, A.-S.W.-N. and H.E.; Funding Acquisition, B.K.P.
DECLARATION OF INTERESTS
The authors declare no competing interests.
Received: September 4, 2018
Revised: October 26, 2018
Accepted: December 5, 2018
Published: December 27, 2018
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12 Cell Metabolism 29, 1–12, April 2, 2019

 Please cite this article in press as: Wedell-Neergaard et al., Exercise-Induced Changes in Visceral Adipose Tissue Mass Are Regulated by IL-6
Signaling: A Randomized Controlled Trial, Cell Metabolism (2018), https://doi.org/10.1016/j.cmet.2018.12.007

STAR+METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER

Antibodies
Tocilizumab. Recombinant humanized monoclonal Roche Pharma AG RRID: AB_2459656
IL-6 receptor antibody (20 mg/mL)

CONTACT FOR REAGENT AND RESOURCE SHARING

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Helga
Ellingsgaard (helga.ellingsgaard@regionh.dk).

EXPERIMENTAL MODEL AND PARTICIPANT DETAILS

Study Design, Participants, and Setting

In this randomized, placebo-controlled, double blind study, abdominally obese men and women were included. From August 2016
to April 2018 participants were enrolled at the Centre for Physical Activity Research (CFAS) in the Capital Region of Denmark. The
full protocol is published (Christensen et al., 2018). Participants were eligible if they were abdominally obese (waist to height
ratio R 0.5 and/or waist circumference R 88/102 for women/men respectively) (Lean et al., 1995), physically inactive (Garber
et al., 2011; Tremblay et al., 2017) and over the age of 18.
Permitted medication should be taken at a stable dose during the last 4 weeks prior to the randomization and remain stable
throughout the study. Exclusion criteria were diabetes (HbA1c R 48 mmol/mol or fasting glucose R 7.0 mmol/l), ischemic heart
disease, atrial fibrillation, pregnancy, biologic rheumatic drugs, systemic prednisolone or other immunosuppressive treatments, or
health conditions that prevented from participating in the exercise intervention (e.g., severe obesity).
All participants provided written informed consent. The study was approved by the Region Ethical Committee, Copenhagen,
Denmark (H-16018062), reported to the Danish Data Protection Agency (2012-58-0004) and registered at Clinicaltrials.gov as
NCT02901496. The study was conducted in accordance with the guidelines for Good Clinical Practice and the Declaration of Hel-
sinki. The results have been reported in accordance with the CONSORT guidelines.
As originally planned, data collected in this study will be published in manuscripts following this one. Among the secondary out-
comes are changes in cardiac adipose tissue, glycemic control, and gastric emptying. Two participants without available MRI scans
were included in the data analysis.

Sequence Generation and Allocation Concealment

The full description has previously been presented (Christensen et al., 2018). In brief, participants were block-randomized into
1:1:1:1:1 1) no exercise + placebo, 2) no exercise + tocilizumab, 3) exercise + placebo, 4) exercise + tocilizumab or 5) resistance
training + placebo (Figure 1) as parallel groups. Randomized allocation of the participants was based on a computer-generated
list of random numbers. Seven blocks of ten and a final block of five participants were planned. To achieve a balanced sample
size of complete cases in each group, dropouts occurring in the first six blocks were replaced in the subsequent block. Dropouts
in the seventh and eighth blocks were not replaced. The allocation sequence was generated by one researcher (K.K.) and concealed
from all other researchers who took part in the enrollment and assessment of participants. To maintain the blinding, the randomized
sequence was delivered concealed to the laboratory personnel who prepared the infusions and delivered the infusions to researchers
in a concealed manner. All researchers and participants of this study were blinded to the tocilizumab/placebo allocation until all data
were collected and analyzed. As decided before the study start (Christensen et al., 2018) participants allocated to the group resis-
tance training + placebo were not included in the analysis of this present paper.

Interventions
Tocilizumab
All randomized participants received intravenous (i.v.) infusions of saline (placebo) or the IL-6 receptor antibody tocilizumab
(RoActemra, Roche Pharma AG) every four weeks and in total three times (Smolen et al., 2008). Participants were instructed not
to change free-living physical activity and eating habits during the study period.
Exercise Training
The individual training programs were designed based on VO2max. To ensure optimal intensity and compliance, educated personnel
supervised all exercise sessions. The exercise modality was high-intensive interval aerobic exercise, performed on an ergometer

Cell Metabolism 29, 1–12.e1–e3, April 2, 2019 e1

 Please cite this article in press as: Wedell-Neergaard et al., Exercise-Induced Changes in Visceral Adipose Tissue Mass Are Regulated by IL-6
Signaling: A Randomized Controlled Trial, Cell Metabolism (2018), https://doi.org/10.1016/j.cmet.2018.12.007

bike. The exercise intensity was progressively increased over the 12-week training period following a protocol with intensities ranging
from 50%–85% of VO2max (Christensen et al., 2018).

METHOD DETAILS

Full descriptions of tests can be found in the study protocol (Christensen et al., 2018). Here we only describe the tests included in this
paper. In brief, the intervention was preceded by three days of pretesting. The first day included medical examination, maximal
oxygen consumption (VO2 max) test, body composition analysis including dual-energy X-ray absorptiometry scan (DXA) and
anthropometrics. The second day included fasting blood samples, and a VO2 max test. Two VO2 max tests were performed to allow
participants to familiarize with the test. The highest VO2max was used in the analysis and in the planning of training intensity. The third
test day included a MRI scan of the abdomen. After the 12-week intervention, post testing identical to the pretesting was performed.

Anthropometrics and Blood Pressure Measurements

Height was measured with a Holtain stadiometer (Holtain Ltd, Crymych, UK) to the nearest 0.1 cm, and body weight was measured
(ADP calibrated electronic scale) to the nearest 0.1 kg. Body Mass Index (BMI) was calculated as weight in kg divided by the square of
height in meters. Waist circumference was measured at the midpoint between the iliac crest and the rib cage on the mid-axillary line
while hip circumference was measured at the point yielding the maximum circumference over the buttocks to the nearest 0,1 cm.
Resting blood pressure and heart rate were measured with an automated oscillometric device on the upper arm (Omron 705IT,
Omron Corporation, Kyoto, Japan).

Body Composition and Analysis

MRI of the abdomen was performed on a 1,5-Tesla MR scanner (Ingenia, Philips, Eindhoven, the Netherlands) 75 axial images
(DIXON breathhold, Slice 5mm, TE 1.7ms, TR 5.5ms, FOV 400mm, inplane res 1.3x1.7mm, NEX 1, scantime 1:08 min.) from the
top of the Thoracic diaphragm to the upper border of the first sacral vertebra were manually analyzed, by two independent blinded
investigators, and the volume was calculated as the sum of all voxels using the software program MANGO version 2.5 (Multi-Images
Analysis GUI, University of Texas Health Science Center, San Antonio, TX) (Lancaster et al., 1991; Positano et al., 2004). The total
volumes were converted into a mass by using the density of body fat at 37 C (Fidanza et al., 1953). The same investigator analyzed
all MRI scans in a blinded manner. A second investigator reanalyzed a subset of the MRI scans (n = 10) in a blinded manner to test the
reliability of the MRI results. The degree of agreement between the results of the two independent investigators was found to be
excellent (Intra class correlation: 0.96 [95% CI 0.58 to 1.0]; p = 0.005) (Koo and Li, 2016).
DXA (Lunar Prodigy, version 8.8; GE Medical Systems, Madison, WI, USA) was used to assess total body fat mass and lean
body mass.

Physical Fitness
Maximal oxygen consumption (VO2max) was determined during a bicycle ergometer test (Monark Ltd, Varberg, Sweden). After a
5 min warm-up at 70 W, the load was increased by 15 W every minute until exhaustion. Ventilation rate, inspired oxygen and expired
carbon dioxide levels were measured using indirect calorimetric and heart rate was assessed simultaneously throughout the duration
of the test (Quark b2, Cosmed, Rome, Italy). A test was valid if the respiratory exchange ratio > 1.1, a plateau in oxygen intake was
reached or the heart rate was ± 10 heartbeats from the estimated maximal heart rate (Edvardsen et al., 2014).

Free-living Physical Activity and Diet

Self-reported dietary intake was registered over 3 days before, during, and after the intervention. Average daily calorie intake was
calculated using madlog-vita.dk. Free living physical activity was registered using accelerometry (AX3, Newcastle, UK), sampling fre-
quency 100 Hz) over 4 days before, during, and after the intervention. Post hoc re-sampling (to 60 Hz, OmGUI v1.0.0.28) and file con-
version (Actilige v6. 11.6 Actigraph, FL, USA) was performed before the physical activity behavior was classified into sedentary, light,
and moderate, and vigorous physical activity (Propero physical activity data, v.1.7.18, University of Southern Denmark, Odense,
Denmark) (Treuth et al., 2004).

Tocilizumab and Placebo Infusions

The recombinant humanized monoclonal IL-6 receptor antibody tocilizumab (RoActemra, Roche Pharma AG) was diluted in NaCl
0.9% to a total volume of 100 mL and infused over 1 hr at a dose of 8 mg/kg body weight (Smolen et al., 2008). Placebo was
100 mL NaCl 0.9% also infused over 1 hr.

Blood Samples
Fasting blood samples were sampled before and after the intervention and analyzed for glucose, insulin, total cholesterol, HDL, LDL,
triglycerides, immune cells (leukocytes, neutrophils, eosinophils, basophiles, lymphocytes, monocytes), Hs-CRP, hemoglobin and
thrombocytes at the Department of Clinical Biochemistry at Rigshospitalet. Plasma was furthermore analyzed for IL-6, sIL-6 receptor,
IL-18, IFN-g, IL-1b, IL-8, IL-10, TNF-a, leptin, adiponectin and IL-1Ra according to manufacturer’s instructions (V-PLEX; Meso Scale
Discovery, USA). Blood samples collected before and at the end of a 45min exercise bout (one of the first three exercise sessions in

e2 Cell Metabolism 29, 1–12.e1–e3, April 2, 2019

 Please cite this article in press as: Wedell-Neergaard et al., Exercise-Induced Changes in Visceral Adipose Tissue Mass Are Regulated by IL-6
Signaling: A Randomized Controlled Trial, Cell Metabolism (2018), https://doi.org/10.1016/j.cmet.2018.12.007

the 12-week intervention) were analyzed for IL-6 (Meso Scale Discovery), epinephrine and norepinephrine using the 2-CAT radioim-
munoassay (Labor Diagnostika Nord, Germany).

QUANTIFICATION AND STATISTICAL ANALYSIS

Sample Size and Power Considerations

A prespecified sample size and power calculation was performed before enrolling participants based on changes in visceral adipose
tissue mass measured from MRI between the two groups most critical to the null hypothesis (exercise + tocilizumab versus exercise +
placebo): For a two-sample pooled t test of a normal mean difference with a two-sided statistical significance level of 0.05 (p < 0.05),
assuming a common standard deviation of 265 (own unpublished data), a sample size of 14 individuals per group is required to obtain
a power of at least 80% to detect a mean difference between groups of 300 g; with 14 individuals in each group the actual power is
82.2% (Christensen et al., 2018).

Statistical Analysis
All analyses were carried out using Stata software, version 13.1 (StataCorp, TX, USA) and tests were evaluated using a significance
level of a = 0.05. Effects of the intervention on pre-specified outcomes were assessed following the per-protocol design according to
the pre-specified protocol (Christensen et al., 2018). Initial modeling was based on Analysis of Covariance (ANCOVA) performed with
change (post-measure minus pre-measure) as the dependent outcome, with a fixed factor for group (four levels) and applying the
value at baseline as a covariate. For each continuous outcome variable (after performing the overall ANCOVA model with four-group
comparison), tests for means adjusted for covariates (least-squares mean adjusted for baseline variables) were performed obtaining
a p value for the contrasts between groups, mainly Group exercise + placebo versus Group exercise + tocilizumab, independent of
what the overall ANCOVA model indicates (as in Table 2). The calculation of LSM is used when a comparison across groups needs to
be made without the influence from differences between groups in the outcome variable at baseline, thus LSM are means adjusted for
baseline. LSM estimation is often assumed to be closer to reality as LSM tends to correct the imbalance of the randomization (Vickers
and Altman, 2001). All results from the ANCOVA and following the ANCOVA model were expressed as estimates, with 95% CI’s to
represent the precision of the estimates. In Tables S2 and S3, p values for unpaired t test and ANOVA models were reported.

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