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Interleukin-6 contributes to early fasting-induced free fatty acid mobilization in mice

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Contracting muscle releases interleukin-6 (IL-6) enabling the metabolic switch from carbohydrate to fat utilization. Similarly, metabolism is switched during transition from fed to fasting state. Herein, we examined a putative role for IL-6 in the metabolic adaptation to normal fasting. In lean C57BL/6J mice, 6 hours of food withdrawal increased gene transcription levels of IL-6 in skeletal muscle but not in white adipose tissue. Concomitantly, circulating IL-6 and free fatty acid (FFA) levels were significantly increased, whereas respiratory quotient (RQ) was reduced in 6-hour fasted mice. In white adipose tissue, phosphorylation of hormone-sensitive lipase (HSL) was increased upon fasting, indicating increased lipolysis. Intriguingly, fasting-induced increase in circulating IL-6 levels and parallel rise in FFA concentration were absent in obese and glucose intolerant mice. A causative role for IL-6 in the physiological adaptation to fasting was further supported by the fact that fasting-induced increase in circulating FFA levels was significantly blunted in lean IL-6 knockout (KO) and lean C57BL/6J mice treated with neutralizing IL-6 antibody. Consistently, phosphorylation of HSL was significantly reduced in adipose tissue of IL-6 depleted mice. Hence, our findings suggest a novel role for IL-6 in energy supply during early fasting.
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Interleukin-6 contributes to early fasting-induced free fatty acid mobilization
in mice
Stephan Wueest,
1,2
* Flurin Item,
1,2
* Christina N. Boyle,
3
Paulin Jirkof,
4
Nikola Cesarovic,
4
Helga Ellingsgaard,
5
Marianne Böni-Schnetzler,
5
Katharina Timper,
5
Margarete Arras,
4
Marc Y. Donath,
5
Thomas A. Lutz,
3,6,7
Eugen J. Schoenle,
1,2
and Daniel Konrad
1,2,6
1
Department of Pediatric Endocrinology and Diabetology and
2
Children’s Research Center, University Children’s Hospital,
Zurich, Switzerland;
3
Institute of Veterinary Physiology, University of Zurich, Zurich, Switzerland;
4
Division of Surgical
Research, University Hospital Zurich, Zurich Switzerland;
5
Division of Endocrinology, Diabetes & Metabolism and
Department of Biomedicine, University Hospital and University of Basel, Basel, Switzerland;
6
Zurich Center for Integrative
Human Physiology, University of Zurich, Zurich, Switzerland; and
7
Institute of Laboratory Animal Science, University of
Zurich, Zurich, Switzerland
Submitted 2 December 2013; accepted in final form 27 March 2014
Wueest S, Item F, Boyle CN, Jirkof P, Cesarovic N, Ellings-
gaard H, Böni-Schnetzler M, Timper K, Arras M, Donath MY,
Lutz TA, Schoenle EJ, Konrad D. Interleukin-6 contributes to early
fasting-induced free fatty acid mobilization in mice. Am J Physiol
Regul Integr Comp Physiol 306: R861–R867, 2014. First published
April 2, 2014; doi:10.1152/ajpregu.00533.2013.—Contracting muscle
releases interleukin-6 (IL-6) enabling the metabolic switch from
carbohydrate to fat utilization. Similarly, metabolism is switched
during transition from fed to fasting state. Herein, we examined a
putative role for IL-6 in the metabolic adaptation to normal fasting. In
lean C57BL/6J mice,6hoffood withdrawal increased gene transcrip-
tion levels of IL-6 in skeletal muscle but not in white adipose tissue.
Concomitantly, circulating IL-6 and free fatty acid (FFA) levels were
significantly increased, whereas respiratory quotient (RQ) was re-
duced in 6-h fasted mice. In white adipose tissue, phosphorylation of
hormone-sensitive lipase (HSL) was increased on fasting, indicating
increased lipolysis. Intriguingly, fasting-induced increase in circulat-
ing IL-6 levels and parallel rise in FFA concentration were absent in
obese and glucose-intolerant mice. A causative role for IL-6 in the
physiological adaptation to fasting was further supported by the fact
that fasting-induced increase in circulating FFA levels was signifi-
cantly blunted in lean IL-6 knockout (KO) and lean C57BL/6J mice
treated with neutralizing IL-6 antibody. Consistently, phosphorylation
of HSL was significantly reduced in adipose tissue of IL-6-depleted
mice. Hence, our findings suggest a novel role for IL-6 in energy
supply during early fasting.
metabolic adaptation; lipolysis; energy supply
WHILE MOST TISSUES rely on glucose as energy substrate in the
postprandial state, energy production from free fatty acid
(FFA) becomes increasingly important upon fasting (7, 26).
Increased fat oxidation reduces the need for glucose (from
glycogen stores or gluconeogenesis) as an energy source.
Moreover, fasting leads to increased production of ketone
bodies in the liver (mainly in the form of -hydroxybutyrate),
which can serve as alternative energy source (7, 9, 20). During
fasting, FFAs are increasingly released from white adipose
tissue, mainly as a consequence of reduced circulating insulin
levels, to comply with the increased demand of liver and other
organs (23). Mechanistically, reduced insulin levels increase
protein kinase A-dependent phosphorylation of hormone-sen-
sitive lipase (HSL) in white adipose tissue, leading to increased
lipolysis (12).
Similar to the adaptation to fasting, increased FFA mobilization
is crucial during prolonged exercise (3). During physical activity,
catecholamine and muscle-derived interleukin 6 (IL-6) stimulate
adipocyte lipolysis, which in turn ensures adequate FFA supply
(14). Indeed, IL-6 alone can induce lipolysis in vivo and in vitro
(17). Moreover, indirect calorimetry in IL-6 KO mice revealed
an elevated respiratory quotient (RQ) suggesting increased
reliance on glucose oxidation for energy production compa-
red with wild-type (WT) mice (8). Hence, IL-6 might be
required for the “metabolic switch” of fuel utilization, from
predominantly carbohydrate to more lipid oxidation. Support-
ing this notion overnight fasting increases circulating IL-6
plasma levels significantly in humans (29). Moreover, such rise
in IL-6 concentration is accompanied by lower RQ (29).
However, the impact of IL-6 on glucose homeostasis and
insulin sensitivity remains unclear (2, 5). On one hand, in-
creased IL-6 action may deteriorate (hepatic) insulin sensitivity
and, thus, contribute to obesity-associated insulin resistance (2,
5, 19, 27). On the other hand, recent reports suggest a role for
hepatic IL-6 signaling in limiting hepatic inflammation,
thereby providing a protective mechanism against local and
systemic insulin resistance (28).
In the present study, we hypothesized that fasting-mediated
release of IL-6 stimulates FFA mobilization via activation of
adipose tissue lipolysis, similar to IL-6 action during exercise,
thereby supporting the fasting-induced metabolic switch from
carbohydrate to lipid oxidation. To test this hypothesis, muscle
IL-6 mRNA levels as well as circulating IL-6 and FFA con-
centrations were determined in fed versus fasted control mice
and in IL-6-deficient mice. Our findings provide evidence for a
novel role of IL-6 in energy supply during early fasting.
MATERIALS AND METHODS
Animals. Male C57BL/6J (C57BL/6JOlaHsd)-mice were pur-
chased from Harlan (AD Horst, The Netherlands), male IL-6 knock-
out (KO), and respective WT mice were obtained from Charles River
Laboratories (Wilmington, MA). All mice were housed in a specific
pathogen-free environment on a 12-h light-dark cycle (light on from
7 PM to 7 AM) and fed ad libitum with regular chow diet (Provimi
Kliba, Kaiseraugst, Switzerland) or high-fat diet (HFD) (D12331,
Research Diets, New Brunswick, NJ). All protocols conformed to the
* S.W. and F.I. contributed equally to this study.
Address for reprint requests and other correspondence: D. Konrad, Univ.
Children’s Hospital, Dept. of Endocrinology and Diabetology, Steinwiesstrasse
75, CH-8032 Zurich (e-mail: daniel.konrad@kispi.uzh.ch).
Am J Physiol Regul Integr Comp Physiol 306: R861–R867, 2014.
First published April 2, 2014; doi:10.1152/ajpregu.00533.2013.
0363-6119/14 Copyright ©2014 the American Physiological Societyhttp://www.ajpregu.org R861
Swiss animal protection laws and were approved by the Cantonal
Veterinary Office in Zurich, Switzerland.
Intraperitoneal glucose tolerance tests. Glucose was injected in-
traperitoneally (2 mg/g body wt) in overnight-fasted mice (n6 mice
per group). Blood glucose concentration was measured in blood from
tail-tip bleedings using a glucometer (AccuCheck Aviva, Roche
Diagnostics, Rotkreuz, Switzerland) as described (19).
Determination of plasma insulin, FFA, ketone body, IL-6, KC, and
TNF-
levels. Plasma insulin and FFA levels were determined as
described (11). Of note, FFA levels were analyzed in plasma sampled
from heart blood. Plasma IL-6, KC (cytokine-induced neutrophil-
attracting chemokine), and tumor necrosis factor-(TNF-) levels
were measured with mouse LINCOplex kits from Linco Research
(Labodia, Yens, Switzerland) and mouse Procarta Cytokine Assay Kit
(Labodia). Blood ketone concentration was determined with the Pre-
cision Xtr ketone meter (Abbott Laboratories, Baar, Switzerland)
allowing measurements with accuracy of one digit after the decimal
point.
IL-6 neutralization. Neutralizing anti-IL-6 (0.5 mg) or an IgG
control antibodies (R&D Systems) were injected intraperitoneally 1 h
before the beginning of the fasting period (n8 or 10 mice per
group).
Metabolic cage analysis. Food and water intake, O
2
consumption,
and CO
2
production were determined for single-housed mice in a
metabolic and behavioral monitoring system (PhenoMaster, TSE
Systems, Bad Homburg, Germany). Mice were given at least 4 days
to acclimate to single caging before experiments were started (n5
to 8 mice per group).
Activity analysis. To test the effects of food deprivation/removal on
general activity, all animals were single housed in observation cages
[type 3 clear-transparent plastic cages (425 mm 266 mm 155
mm)] without cage grids; animals were provided with unrestricted
access to food and drinking water, sawdust bedding, one red mouse
house as shelter (Indulab, Gams, Switzerland), and one Nestlet
(5 cm 5 cm) consisting of cotton fibers (Indulab) as nesting material
for 3 days before observation. Behavior was digitally recorded on day
4. For recording of baseline activity, animals were fed ad libitum and
observed for 24 h; subsequent on day 5 animals were observed for 24
h under the same conditions but without access to food (deprivation).
The recorded 24-h video sequences were analyzed using EthovisionX
software (Noldus, Wageningen, The Netherlands). As a parameter of
activity, distance moved in centimeters was recorded (n8 mice per
group).
RNA extraction and quantitative reverse transcription-PCR. Total
RNA from quadriceps muscle, epididymal white adipose tissue, liver,
and brain was extracted with the RNeasy lipid tissue mini kit (Qiagen,
Basel, Switzerland). RNA was reverse transcribed with Superscript III
Reverse Transcriptase (Invitrogen, Basel, Switzerland) using random
hexamer primer (Invitrogen). Taqman system (Applied Biosystems,
Rotkreuz, Switzerland) was used for real-time PCR amplification.
Relative gene expression was obtained after normalization to 18S
RNA (Applied Biosystems), using the formula 2
⫺⌬⌬cp
(18). The gene
expression assays used were the following: IL-6, Mm00446190_m1;
CPT-1, Mm00550438_m1; and PGC-1, Mm01208835_m1 (Applied
Biosystems).
Muscle glycogen assay. Skeletal muscle of 10 –20 mg was placed
in duplicates in microfuge tubes with 500 l 2 M HCl. Tubes were
boiled for 2 h and reconstituted to original volume with ddH
2
O. Five
hundred microliters of 2 M NaOH were added for neutralization of the
acid. Tubes were vortexed to break up muscle tissue. One hundred
microliters of standard (Sigma, Buchs, Switzerland), ddH
2
O (blank),
or sample were mixed with 1 ml of hexokinase reagent (Sigma) and
incubated for 10 min at room temperature. Samples and standards
were finally read in a spectrophometer at 340 nm (n10 chow-fed
and n3 HFD-fed mice per group).
Western blot. Tissues were lysed and Western blots were per-
formed as previously described (27). Membranes were blocked for 1
h in 5% nonfat dry milk (Bio-Rad) and incubated overnight at 4°C on
a rocking platform with respective primary antibodies diluted 1:1,000.
Primary antibodies used were the following: anti-phospho-p38, anti-
phospho-HSL (Ser660) (both from Cell Signaling, Danvers, MA), and
anti-actin (Millipore, Zug, Switzerland).
Data analysis. Data are presented as means SE and were
analyzed by unpaired Student’s t-test or ANOVA with Bonferroni-
corrected post hoc tests. Log transformation was performed to obtain
normally distributed data where necessary.
0
500
1000
1500
2000
2500
8 am 2 pm 8 pm 8 am
*
Time of the day
Plas ma IL- 6 (p g/ml)
Fasted
Fed
*
0
100
200
300
400
500
8 am 2 pm 8 pm 8 am
***
*
Time of the day
Plasma insulin (pmol/l)
Fasted
Fed
0
5
10
15
8 am 2 pm 8 pm 8 am
Time of the day
Blood glucose (mmol/l)
Fasted
Fed
***
0.0
0.5
1.0
1.5
2.0
2.5
8 am 2 pm 8 pm 8 am
Time of the day
Blood ketone (mmol/l)
Fasted
Fed
AB
CD
Fig. 1. Increased circulating IL-6 levels after
6 h of fasting. Chow-fed C57BL/6J mice
were either fasted starting at 8.00 AM or fed
ad libitum, and blood was sampled by tail tip
bleeding at indicated time points. Shown are
values for ketone bodies (-hydroxybutyrate)
(A), blood glucose (B), IL-6 (C), and plasma
insulin (D). Results are means SE; n
4 6 mice. Measured parameters were signif-
icantly different between the groups for time
(B:P0.001, C:P0.01 and D:P
0.05), and there were time group interac-
tions (B:P0.001, C:P0.07 and D:P
0.001) (ANOVA). *P0.05, **P0.01,
***P0.001 (Bonferroni-corrected post
hoc tests). Of note, data for ketone body
measurements (A) were not normally distrib-
uted (probably due to measurement accuracy;
see MATERIALS AND METHODS), and, hence,
ANOVA could not be performed.
R862 IL-6-MEDIATED RELEASE OF FREE FATTY ACIDS IN EARLY FASTING
AJP-Regul Integr Comp Physiol doi:10.1152/ajpregu.00533.2013 www.ajpregu.org
RESULTS
Plasma IL-6 levels are increased upon fasting in chow-fed
C57BL/6J mice. To examine the potential role of IL-6 in
metabolic adaptation to fasting, circulating IL-6 levels were
assessed in mice after food withdrawal. Three-month-old
C57BL/6J mice were either randomly fed or fasted for 24 h
(starting at 8 am), and blood was sampled after 6, 12, and 24
h. As expected, fasting induced an increase in blood ketone
levels, with an increase already after fasting for 6 h, whereas
blood glucose levels were only significantly different after 24
h of fasting (Fig. 1B). In parallel, plasma IL-6 levels increased
3-fold after 6 h and 4.5-fold after 12 h of fasting (Fig. 1C). In
contrast, there was no fasting-induced increase in the concen-
tration of other circulating cytokines such as keratinocyte
chemoattractant (KC, the mouse homologue of interleukin-8)
and TNF-(data not shown), suggesting that the elevation in
circulating IL-6 in response to fasting does not indicate acti-
vation of classical pro-inflammatory cytokine cascades. Of
note, circulating insulin levels were not significantly different
between mice fasted for 6 h and random fed mice, whereas
they were more than fivefold decreased in mice fasted for 12 h
(Fig. 1D). Since insulin is a major regulator of circulating FFA
CPT-1 PGC-1α
0
1
2
3
*
Sk. muscle mR NA
(relat ive to 18s)
*
Fed
Fasted
Fed Fasted
0.0
0.2
0.4
0.6
0.8
1.0
RQ
**
Fed Fasted
0
1
2
3
4
**
WAT pHSL (Ser660)
(relative to actin)
Fed Fasted
0.0
0.2
0.4
0.6
0.8
Plasma FFA (mmol/l)
**
AB
CD
pHSL
Actin
Fig. 2. Increased free fatty acid (FFA) levels
after6hoffasting. Chow-fed C57BL/6J
mice were either fasted (open bars) starting at
8.00 AM or fed ad libitum (closed bars).
A: plasma FFA levels (n4 mice) after 6 h.
B: representative Western blots of epididy-
mal adipose tissue of fed and fasted mice.
Graph depicts results of 7 mice per group.
C: respiratory quotient (RQ) was determined
in metabolic cages in mice fed ad libitum or
in mice fasted for 6 h. Shown are average RQ
data recorded during the last hour of the
experiment (n6 – 8 mice). D: skeletal mus-
cle mRNA expression of carnitine palmitoyl-
transferase 1 (CPT-1) and peroxisome prolif-
erator-activated receptor gamma coactivator
1-(PGC-1
) was analyzed in fed and fasted
mice and normalized to 18S RNA. All results
are means SE; n4 mice. *P0.05,
**P0.01 (Student’s t-test).
Sk. muscle WAT Liver Brain
0.0
0.5
1.0
1.5
2.0
2.5
IL-6 mRNA
(relative to 18s)
** Fed
Fasted
**
Fed Fasted
0
1
2
3
4
*
Sk. muscle p-p38 MAPK
(relat ive to actin)
p-p38
Actin
Fed Fasted
0
50
100
150
200
250
Distance moved (m)
Fed Fasted
0
5
10
15
20
25
**
Sk. muscle glycogen
(
μ
mol glucose/g tissue)
D
AB
C
Fig. 3. Increased IL-6 mRNA expression in
skeletal muscle after6hoffasting. Chow-fed
C57BL/6J mice were either fasted at 8.00 AM
or fed ad libitum. After 6 h, mice were eutha-
nized, and quadriceps muscle, epididymal white
adipose tissue, liver as well as brain were re-
moved. A: total RNA was extracted from tissue
and quantitative RT-PCR was performed. The
level of IL-6 mRNA expression was normal-
ized to 18S RNA and shown relative to fed
mice. n3– 6 mice. B: glycogen content was
determined in quadriceps muscle with a
hexokinase assay (as described in MATERIALS
AND METHODS). n10 mice. C: representa-
tive Western blot of total muscle lysates of
fed and fasted mice. Graphs show results of 4
mice. D: chow-fed C57BL/6J mice were ei-
ther fasted at 8.00 AM or fed ad libitum, and
locomotor activity was analyzed during 6 h.
Shown are values for moved distances in
meters. All results are the means SE; n
8 mice. *P0.05, **P0.01 (Student’s
t-test).
R863IL-6-MEDIATED RELEASE OF FREE FATTY ACIDS IN EARLY FASTING
AJP-Regul Integr Comp Physiol doi:10.1152/ajpregu.00533.2013 www.ajpregu.org
levels by inhibiting lipolysis, which results in decreased FFA
concentrations, we focused our additional studies on mice
fasted for 6 h, which does not significantly affect blood insulin
levels. To further investigate whether6hoffasting affect
metabolism in lean mice, FFA levels were analyzed. As shown
in Fig. 2A, plasma FFA levels were significantly increased in
mice fasted for 6 h. Consistently, phosphorylation of HSL was
significantly increased in white adipose tissue of fasted mice
(Fig. 2B), indicating increased lipolysis. Moreover,6hof
fasting led to a significant reduction in the RQ, suggesting
increased fat oxidation (Fig. 2C). In agreement, mRNA expres-
sion of carnitine palmitoyltransferase 1 (CPT-1) and peroxi-
some proliferator-activated receptor gamma coactivator 1-
(PGC-1
) [two enzymes involved in fat oxidation (21)] were
significantly increased in skeletal muscle of fasted mice (Fig.
2D). Of note, body weight was similar in fed and fasted mice
(27.6 0.8 g vs. 27.1 0.9 g) and randomly fed mice ate on
average 0.3 0.1 g during the 6-h period.
To determine the source of IL-6 production during fasting,
its mRNA expression was assessed in white adipose tissue and
skeletal muscle, the two major sources of circulating IL-6
levels (5, 14) as well as in the liver and brain. As depicted in
Fig. 3A,6hoffasting upregulated IL-6 mRNA expression in
skeletal muscle, but not in white adipose tissue. Of note, IL-6
transcription was decreased by6hoffasting in the liver and
brain (Fig. 3A). It was previously suggested that intramuscular
glycogen content is an important enhancer of IL-6 mRNA
expression in skeletal muscle during exercise (14). We there-
fore hypothesized that a fasting-induced decrease in muscle
glycogen content might trigger IL-6 expression in skeletal
muscle. As shown in Fig. 3B,6hoffasting reduced glycogen
levels by 25% in skeletal muscle of chow-fed C57BL/6J
mice paralleling the rise in muscle IL-6 mRNA levels (Fig.
3A). In addition, fasting increased phosphorylation of the p38
mitogen-activated protein kinase (p38 MAPK) in skeletal mus-
cle (Fig. 3C), a stress kinase involved in skeletal muscle IL-6
expression (16). Of note, the decrease in skeletal muscle
glycogen content could not be attributed to increased locomo-
tor activity in response to fasting (Fig. 3D). This finding
suggests that the observed increase in IL-6 mRNA levels upon
fasting was not due to increased physical activity/muscle con-
traction as part of increased food-seeking behavior, and thus,
differs from IL-6 induction in muscle in response to exercise.
Loss of fasting-induced regulation of IL-6 and FFA levels in
HFD-fed mice. Obese and glucose-intolerant mice have a
blunted metabolic adaptation to fasting (24). To investigate
whether fasting-induced IL-6 is disrupted in mice with im-
paired metabolic flexibility, C57BL/6J mice were fed a high-
fat diet (HFD) for 6 wk. As expected, HFD increased body
weight (28.4 0.4 g chow-fed vs. 32.6 0.4 g HFD, P
0.01), impaired glucose tolerance (Fig. 4A), and induced insu-
lin resistance [fasting insulin levels: 76.0 4.4 pmol/l chow-
HFD Fed HFD Fasted
0
10
20
30
40
50
Plasma IL-6 (pg/ml)
HFD Fed HFD Fasted
0
20
40
60
Sk. muscle glycogen
(
μ
mol glucose/g tissue)
HFD Fed HFD Fasted
0.0
0.5
1.0
1.5
Sk. muscle IL-6 mRNA
(relative to fed mice)
030 60 90 120
0
10
20
30
****
**
**
**
HFD
Chow
Time (min)
Blood glucose
(mmol/ l)
D
AB
C
HFD Fed HFD Fasted
0.0
0.5
1.0
1.5
Plasma FFA (mmol/l)
E
Fig. 4. Loss of fasting-induced increase in
IL-6 plasma levels in high-fat diet-fed mice.
A: intraperitoneal glucose tolerance tests in
chow-fed and HFD-fed C57BL/6J mice.
Chow-fed mice were the same animals as
used for blood sampling in Fig. 1. n6
mice. HFD-fed mice were either fasted (open
bars) starting at 8.00 AM or fed ad libitum
(closed bars) for 6 h. Shown are plasma IL-6
concentrations (n6 mice) (B), skeletal mus-
cle IL-6 mRNA expression (n4 mice) (C),
skeletal muscle glycogen (n3 mice) (D), and
plasma FFA levels (n4 –5 mice) (E). All
results are means SE. Glucose excursion (A)
was significantly different between the groups
for time (P0.001), and there was a
timegroup interaction (P0.001; ANOVA).
*P0.05, **P0.01, ***P0.001 Bon-
ferroni-corrected post hoc tests.
R864 IL-6-MEDIATED RELEASE OF FREE FATTY ACIDS IN EARLY FASTING
AJP-Regul Integr Comp Physiol doi:10.1152/ajpregu.00533.2013 www.ajpregu.org
fed vs. 151.5 19.7 pmol/l HFD, P0.01; homeostatic
model assessment of insulin resistance (HOMA-IR): 2.2 0.2
chow-fed vs. 5.5 0.7 HFD, P0.01] compared with
chow-fed mice that showed elevated IL-6 levels upon fasting
(Fig. 1). Interestingly,6hoffasting had no impact on circu-
lating IL-6 levels in HFD-fed mice (Fig. 4B). Consistent with
similar circulating IL-6 levels in fed and fasted HFD mice,
IL-6 mRNA expression in skeletal muscle (Fig. 4C) as well as
skeletal muscle glycogen levels (Fig. 4D) were not different
between fed and fasted mice under HFD. Concomitantly, there
was no increase in fasting-induced FFA concentration in obese
and glucose-intolerant mice (Fig. 4E). Of note, FFA levels
were markedly higher than in lean chow-fed mice (Fig. 2B).
Thus IL-6 may contribute to early fasting-induced metabolic
adaptations in lean but not obese, glucose-intolerant mice.
Fasting-induced increase in FFA levels is reduced in lean
IL-6 KO mice and in lean mice injected with neutralizing IL-6
antibody. IL-6 KO mice were used to further assess a causative
contribution of IL-6 in fasting-induced increase in circulating
FFA levels. Glucose tolerance was not different in 3-mo-old
chow-fed IL-6 KO and WT mice (Fig. 5A), confirming previ-
ous findings in young IL-6 KO mice (6). In addition, there was
no difference in blood glucose levels between the two groups
in fed and fasted mice (Fig. 5B). Importantly, fasting-induced
increase in FFA levels was significantly blunted in IL-6 KO
mice compared with WT mice (Fig. 5C), whereas no difference
in plasma insulin levels was observed (Fig. 5D).
Since the absence of IL-6 during development might have
led to (metabolic) (mal)adaptation in IL-6 KO mice, a second
approach was used to study the potential role of acute IL-6
depletion in early FFA mobilization. Chow-fed C57BL/6J
mice were treated either with a neutralizing IL-6 (nIL-6) or an
isotype control (IgG) antibody and subsequently fasted for 6 h.
While there was no difference in blood glucose concentration
between the two groups after6hoffasting (Fig. 6A), FFA
levels were significantly lower in mice injected with nIL-6
antibody compared with IgG-injected mice (Fig. 6B). Of note,
white adipose tissue of fasted mice treated with nIL-6 antibody
revealed significantly reduced phosphorylation of HSL, sug-
gesting blunted lipolysis (Fig. 6C). Importantly, insulin levels
were similar in the two fasted groups (Fig. 6D). Moreover,
neutralization of IL-6 did not alter RQ and mRNA expression
of CPT-1 and PGC-1
in skeletal muscle (Fig. 6, Eand F)
suggesting that muscle lipid oxidation is not affected by IL-6
neutralization. In summary, experiments in IL-6-depleted mice
further confirm the notion that the fasting-induced rise in
circulating FFA levels is IL-6 dependent.
DISCUSSION
In the present study we identified a role for IL-6 in the
fasting-induced increase in circulating FFA levels. The major
findings of this study supporting this proposition are 1) fasting
increases circulating IL-6 levels in lean mice; 2) depletion
of IL-6 (either by IL-6 KO or by neutralization of circulating
IL-6) blunts the fasting-induced rise in circulating FFA levels
in lean mice; and 3) obese and glucose-intolerant mice lack the
fasting-induced increase in circulating IL-6 and FFA levels.
What is the source of the fasting-induced increase in circu-
lating IL-6 levels? IL-6 mRNA expression was increased in
skeletal muscle but not in white adipose tissue, liver, and brain
6 h after fasting. Since both skeletal muscle and adipose tissue
contribute significantly to circulating IL-6 levels at rest (5, 14),
our data suggest that skeletal muscle is the main contributor to
increased circulating IL-6 levels during early fasting. Compat-
ible with such notion, fasting increased phosphorylation of p38
MAPK in skeletal muscle, which was previously found to
contribute to IL-6 transcription and secretion (16). Although
the link is only associative, IL-6 transcription may be triggered
by decreased muscle glycogen levels, as was previously shown
for physical activity (14). However, in contrast to physical
activity, decreased glycogen levels during fasting does not
030 60 90 120
0
10
20
30
Time (min)
Blood glucose
(mmol/l)
WT IL-6 KO
0.0
0.2
0.4
0.6 **
*
Plasma FFA (mmol/l)
WT IL-6 KO
0
50
100
150
200
Plasma insulin (pmol/l)
WT IL-6 KO
0
5
10
15
Blood glucose
(mmol/l)
AB
CD
Fig. 5. Fasting-induced increase in FFA lev-
els is reduced in IL-6 KO mice. A: intraperi-
toneal glucose tolerance tests in chow-fed
WT (Œ) and IL-6 KO () mice. n5– 6
mice. Chow-fed WT and IL-6 KO mice were
either fasted (open bars) starting at 8.00 AM
or fed ad libitum (closed bars) for 6 h. Shown
are blood glucose (n5 mice) (B), plasma
FFA (n4 –5) (C), and plasma insulin
levels (n4 –5 mice) (D). All results are
means SE. *P0.05, **P0.01 (Stu-
dent’s t-test).
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AJP-Regul Integr Comp Physiol doi:10.1152/ajpregu.00533.2013 www.ajpregu.org
seem to be the consequence of enhanced locomotor activity,
e.g., due to food-seeking behavior. Regardless of the mecha-
nism for fasting-induced induction of IL-6 in skeletal muscle,
from a physiological point of view it would seem “logical” that
skeletal muscle as the major fuel consumer would signal the
need to mobilize fat stores for fatty acid-based energy gener-
ation, using IL-6 as a “second messenger.” In parallel to the
activation of lipolysis in white adipose tissue via endocrine
signaling, increased expression of IL-6 in skeletal muscle may
impact on local FFA release, since IL-6 was shown to stimulate
lipolysis in skeletal muscle (13). Moreover, although we found
no difference in mRNA expression of CPT-1 and PGC-1
in
skeletal muscle after IL-6 neutralization, we cannot rule out a
direct effect of fasting-induced IL-6 on fatty acid oxidation in
skeletal muscle (4). Of note, observed differences in circulating
IL-6 levels in ad libitum-fed lean WT mice at different time
points may be explained by a circadian dependency of IL-6
secretion (25). Alternatively, stress (induced by blood sam-
pling) may induce an epinephrine-mediated release of IL-6
(14). Whereas overnight fasting in humans increased circulat-
ing IL-6 levels (29), intermittent fasting even decreased IL-6 in
the circulation (1). In mice, overnight fasting had no effect on
basal IL-6 levels, but it increased exercise-induced circulating
IL-6 (15). Hence, the effect of food deprivation on circulating
IL-6 levels may depend on time of day, duration, as well as the
pattern of fasting (single bout vs. intermittent).
The finding of decreased IL-6 expression in brain and liver
not only demonstrates the unique role of skeletal muscle IL-6
in metabolic adaptation, but also highlights a possible regula-
tory role for IL-6 in the adaptation to short-term fasting also in
these tissues: IL-6 was shown to have anorexigenic effects in
the brain (22) and thus decreased local IL-6 production during
fasting would support food-seeking behavior. Complementa-
rily, IL-6 decreases hepatic gluconeogenesis (10). Therefore,
its decreased hepatic expression would support the required
upregulation of hepatic glucose production during fasting.
Insulin is a major regulator of circulating FFA levels by
inhibiting lipolysis in white adipose tissue. As pointed out, we
focused our studies on mice fasted for 6 h, which did not
significantly affect blood insulin levels. Nevertheless, the ob-
served slight reduction in circulating insulin levels upon6hof
fasting may still have affected circulating FFA levels in lean WT
mice. However, circulating insulin levels were not increased in
fasted IL-6 KO mice and in mice treated with nIL-6 antibody
compared with their respective fasted control mice. Such result
would suggest that decreased circulating FFA levels in IL-6-
depleted mice after6hoffasting were not dependent on increased
circulating insulin levels. In addition, decreased phosphorylation
Fasted IgG Fasted nIL-6
0
50
100
150
200
250
Plasma insulin (pmol/l)
Fasted IgG Fasted nIL-6
0.0
0.5
1.0
1.5
*
WAT pHSL (Ser660)
(relative to actin)
Fasted IgG Fasted nIL-6
0
5
10
15
Blood glucose
(mmol/l)
Fasted IgG Fasted nIL-6
0.0
0.2
0.4
0.6
0.8
**
Plasma FFA (mmol/l)
pHSL
Actin
AB
CD
Fasted IgG Fasted nIL-6
0.0
0.2
0.4
0.6
0.8
1.0
RQ
CPT-1 PGC-1α
0
1
2
Sk. muscle mRNA
(relative to 18s)
EF
Fig. 6. Fasting-induced increase in FFA lev-
els is reduced in mice treated with neutraliz-
ing IL-6 antibody. Chow-fed WT mice were
treated with either neutralizing IL-6 (hatched
bars) or control IgG (open bars) antibody and
fasted for 6 h starting at 8.00 AM. Shown are
blood glucose levels (n4 mice) (A) and
plasma FFA levels (n8 –10 mice) (B) after
6 h of fasting. C: representative Western
blots of epididymal adipose tissue of fasted
mice. Graph depicts results of 3 mice per
group. D: plasma insulin levels (n8 –10
mice) after6hoffasting. E: respiratory
quotient (RQ) was determined in metabolic
cages in mice fed ad libitum or in mice fasted
for 6 h. Shown are average RQ data recorded
during the last hour of the experiment (n
5–7 mice). F: skeletal muscle mRNA expres-
sion of CPT-1 and PGC-1was analyzed in
fed and fasted mice and normalized to 18S
RNA. All results are means SE; n4–6
mice. *P0.05, **P0.01 (Student’s
t-test).
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AJP-Regul Integr Comp Physiol doi:10.1152/ajpregu.00533.2013 www.ajpregu.org
of HSL in white adipose tissue of IL-6-depleted mice upon fasting
suggests that IL-6 induces lipolysis in adipose tissue and thereby
contributes to the fasting-induced increase in circulating FFA
levels. Our data are in agreement with a previous study reporting
a lipolytic effect of IL-6 in adipocytes (17).
Obese and glucose-intolerant mice have a blunted metabolic
adaptation to fasting (24). Such impaired metabolic flexibility,
recently recognized as a potentially highly clinically relevant
early characteristic of individuals suffering from obesity or
glucose intolerance, may in fact be due to a failure to mount the
normal rise in IL-6 levels in response to fasting.
Perspectives and Significance
Our results indicate a novel physiological role for IL-6 in
early fasting-induced increase in circulating FFA levels and,
thus, metabolic adaptation. Moreover, they suggest that an
impaired rise of IL-6 in response to fasting may contribute to
constrained metabolic flexibility characteristic for obesity-
associated glucose intolerance.
ACKNOWLEDGMENTS
We thank Ramona Meyer from Abbott Laboratories for providing Precision
Xtra ketone test stripes to measure blood ketone concentration and Prof. Dr.
Giatgen Spinas for continuous support.
GRANTS
This work was supported by grants from the Swiss National Science
Foundation (310030-124729), the European Foundation for the Study of
Diabetes (both to D. Konrad) and the Foundation for Research at the Medical
Faculty, University of Zurich (to F Item).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: S.W., M.A., M.Y.D., T.A.L., and D.K. conception and
design of research; S.W., F.I., C.N.B., P.J., N.C., H.E., M.B.-S., and K.T.
performed experiments; S.W., F.I., C.N.B., P.J., N.C., H.E., M.B.-S., K.T., M.A.,
M.Y.D., T.A.L., and D.K. analyzed data; S.W., F.I., E.J.S., and D.K. interpreted
results of experiments; S.W., F.I., and D.K. prepared figures; S.W. and D.K.
drafted manuscript; S.W., F.I., M.A., M.Y.D., T.A.L., E.J.S., and D.K. edited and
revised manuscript; S.W., F.I., C.N.B., P.J., N.C., H.E., M.B.-S., K.T., M.A.,
M.Y.D., T.A.L., E.J.S., and D.K. approved final version of manuscript.
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