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


Abstract and Figures

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,
* Flurin Item,
* Christina N. Boyle,
Paulin Jirkof,
Nikola Cesarovic,
Helga Ellingsgaard,
Marianne Böni-Schnetzler,
Katharina Timper,
Margarete Arras,
Marc Y. Donath,
Thomas A. Lutz,
Eugen J. Schoenle,
and Daniel Konrad
Department of Pediatric Endocrinology and Diabetology and
Children’s Research Center, University Children’s Hospital,
Zurich, Switzerland;
Institute of Veterinary Physiology, University of Zurich, Zurich, Switzerland;
Division of Surgical
Research, University Hospital Zurich, Zurich Switzerland;
Division of Endocrinology, Diabetes & Metabolism and
Department of Biomedicine, University Hospital and University of Basel, Basel, Switzerland;
Zurich Center for Integrative
Human Physiology, University of Zurich, Zurich, Switzerland; and
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.
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:
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 Society 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
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
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
Metabolic cage analysis. Food and water intake, O
and CO
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
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
(18). The gene
expression assays used were the following: IL-6, Mm00446190_m1;
CPT-1, Mm00550438_m1; and PGC-1, Mm01208835_m1 (Applied
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
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
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.
8 am 2 pm 8 pm 8 am
Time of the day
Plas ma IL- 6 (p g/ml)
8 am 2 pm 8 pm 8 am
Time of the day
Plasma insulin (pmol/l)
8 am 2 pm 8 pm 8 am
Time of the day
Blood glucose (mmol/l)
8 am 2 pm 8 pm 8 am
Time of the day
Blood ketone (mmol/l)
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.
AJP-Regul Integr Comp Physiol doi:10.1152/ajpregu.00533.2013
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
Sk. muscle mR NA
(relat ive to 18s)
Fed Fasted
Fed Fasted
WAT pHSL (Ser660)
(relative to actin)
Fed Fasted
Plasma FFA (mmol/l)
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
) 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
(relative to 18s)
** Fed
Fed Fasted
Sk. muscle p-p38 MAPK
(relat ive to actin)
Fed Fasted
Distance moved (m)
Fed Fasted
Sk. muscle glycogen
mol glucose/g tissue)
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
AJP-Regul Integr Comp Physiol doi:10.1152/ajpregu.00533.2013
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-
) [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
Plasma IL-6 (pg/ml)
HFD Fed HFD Fasted
Sk. muscle glycogen
mol glucose/g tissue)
HFD Fed HFD Fasted
Sk. muscle IL-6 mRNA
(relative to fed mice)
030 60 90 120
Time (min)
Blood glucose
(mmol/ l)
HFD Fed HFD Fasted
Plasma FFA (mmol/l)
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.
AJP-Regul Integr Comp Physiol doi:10.1152/ajpregu.00533.2013
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.
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
Time (min)
Blood glucose
0.6 **
Plasma FFA (mmol/l)
Plasma insulin (pmol/l)
Blood glucose
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).
AJP-Regul Integr Comp Physiol doi:10.1152/ajpregu.00533.2013
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
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
Plasma insulin (pmol/l)
Fasted IgG Fasted nIL-6
WAT pHSL (Ser660)
(relative to actin)
Fasted IgG Fasted nIL-6
Blood glucose
Fasted IgG Fasted nIL-6
Plasma FFA (mmol/l)
Fasted IgG Fasted nIL-6
CPT-1 PGC-1α
Sk. muscle mRNA
(relative to 18s)
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
AJP-Regul Integr Comp Physiol doi:10.1152/ajpregu.00533.2013
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.
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.
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).
No conflicts of interest, financial or otherwise, are declared by the author(s).
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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... There is strong evidence, from proteomics studies using in vitro and in vivo models, that many myokines are key endocrine mediators in glucose and lipid metabolism, especially in response to exercise, through crosstalk with other tissues, including WAT. IL-6 is known to increase lipolysis and fatty acid release from WAT [117]; it has been observed that exercise training leading to reduction in VAT was avoided by IL-6 receptor blockade with tocilizumab (IL-6 receptor antibody) [118]. ...
Full-text available
As a result of aging, body composition changes, with a decline in muscle mass and an increase in adipose tissue (AT), which reallocates from subcutaneous to visceral depots and stores ectopically in the liver, heart and muscles. Furthermore, with aging, muscle and AT, both of which have recognized endocrine activity, become dysfunctional and contribute, in the case of positive energy balance, to the development of sarcopenic obesity (SO). SO is defined as the co-existence of excess adiposity and low muscle mass and function, and its prevalence increases with age. SO is strongly associated with greater morbidity and mortality. The pathogenesis of SO is complex and multifactorial. This review focuses mainly on the role of crosstalk between age-related dysfunctional adipose and muscle cells as one of the mechanisms leading to SO. A better understanding of this mechanisms may be useful for development of prevention strategies and treatments aimed at reducing the occurrence of SO.
... Upon targeting adipocytes, IL-6 plays an indirect role in driving HGP process. In response to exercise or early fasting, skeletal muscle-derived IL-6 promotes lipolysis in WAT, thereby providing materials to support HGP (45)(46)(47). Of note, in case of morbid obesity, ATMs produce large amounts of IL-6 to stimulate excess WAT lipolysis, which abrogates insulin action on HGP suppression, resulting in hyperglycemia in rodents or adolescents (48,49). ...
Full-text available
Hepatic glucose production (HGP) is fine-regulated via glycogenolysis or gluconeogenesis to maintain physiological concentration of blood glucose during fasting-feeding cycle. Aberrant HGP leads to hyperglycemia in obesity-associated diabetes. Adipose tissue cooperates with the liver to regulate glycolipid metabolism. During these processes, adipose tissue macrophages (ATMs) change their profiles with various physio-pathological settings, producing diverse effects on HGP. Here, we briefly review the distinct phenotypes of ATMs under different nutrition states including feeding, fasting or overnutrition, and detail their effects on HGP. We discuss several pathways by which ATMs regulate hepatic gluconeogenesis or glycogenolysis, leading to favorable or unfavorable metabolic consequences. Furthermore, we summarize emerging therapeutic targets to correct metabolic disorders in morbid obesity or diabetes based on ATM-HGP axis. This review puts forward the importance and flexibility of ATMs in regulating HGP, proposing ATM-based HGP modulation as a potential therapeutic approach for obesity-associated metabolic dysfunction.
... For example, in fasting state, metabolism was transiently switched from carbohydrate to lipid utilization in humans [89]. In this context, circulating IL-6 level was increased, contributing to TG mobilization and increased oxidation of FFAs in adipose tissue in mice [90]. In fact, exercise-induced IL-6 accelerated lipolysis and 8 ...
Full-text available
Adipose tissue macrophages (ATM) are a major source of low-grade inflammation in obesity, and yet reasons driving ATM accumulation in white adipose tissue (WAT) are not fully understood. Emerging evidence suggested that ATM underwent extensive remodeling in obesity. In addition to abundance, ATM in obesity were lipid-laden and metabolically reprogrammed, which in turn was tightly related to their functional alterations and persistence in obesity. Herein, we aimed to discuss that activation of lipid sensing signaling associated with metabolic reprogramming in ATM was indispensible for their migration, retention, or proliferation in obesity. Likewise, lipolysis also induced similar but transient ATM remodeling. Therefore, we assumed that obesity might share overlapping mechanisms with lipolysis in remodeling ATM. Formation of crown-like structures (CLS) in WAT was presumably a common event initiating ATM remodeling, with a spectrum of lipid metabolites released from adipocytes being potential signaling molecules. Moreover, adipose interlerkin-6 (IL-6) exhibited homologous alterations by obesity and lipolysis. Thus, we postulated a positive feedback loop between ATM and adipocytes via IL-6 signaling backing ATM persistence by comparison of ATM remodeling under obesity and lipolysis. An elucidation of ATM persistence could help to provide novel therapeutic targets for obesity-associated inflammation.
... Physical exercise increases IL-6 levels in the circulation by 100-fold and this results to a constellation of biological changes generally considered antiinflammatory (Pedersen and Febbraio, 2008). Hyperthermia and fasting also induce healthpromoting changes at least in part by increasing the production of IL-6 (Stewart et al., 2010;Wueest et al., 2014). It is noteworthy that in experimental animals, IL-6 administration alone did not produce sickness behaviour, however, IL-6, in presence of IL-1β induced sickness behavior (Lenczowski et al., 1999). ...
Major depressive disorder (MDD) is a common, severe and disabling neuropsychiatric disorder with a heterogenous aetiology. Among the most widely recognized etiological models, immunopathogenesis is a predominant one. Numerous studies have demonstrated aberrant levels of inflammatory markers in the peripheral blood, cerebrospinal fluid (CSF) and brain of patients with MDD. Multiple studies and meta-analyses have reported increased peripheral levels of acute phase proteins, and pro-inflammatory cytokines, particularly IL-1β, TNF-α, and IL-6 in MDD. Post-mortem brain studies similarly demonstrate upregulated expressions of these pro-inflammatory cytokines. This along with evidence of monocytic, lymphocytic and microglial activation, suggest an activated inflammatory response system (IRS) in MDD. A few studies show increased levels of anti-inflammatory cytokines or defective inflammatory pathways and a deficit in T cell maturation and responses in MDD patients. This suggests the presence of a Compensatory Immune Response System (CIRS), which can counterbalance the effects of IRS in major depression. More recently, simultaneously increased levels of both the pro-and anti-inflammatory cytokines are reported in the brain of MDD patients; this indicates activity of both the IRS and CIRS in MDD. The IRS and CIRS are the evolutionarily conserved and integral elements of an overarching system. The relevance of a dysregulated IRS-CIRS system in the neurobiological construct of MDD is just beginning to be understood. Speculation is rife that the disrupted IRS-CIRS elements might determine the onset, episodes, neuroprogressive processes, treatment response as well as recovery of patients with MDD. Notably, the signatures of an activated IRS-CIRS might emerge as potential biomarkers of MDD. Herein, an attempt has been made to highlight the biology and pathobiological relevance of IRS-CIRS activation in MDD and provide an insight into the role of these components in pharmacological therapy.
... In particular, the prototypic inflammatory cytokine tumor necrosis factor (TNF) modulates systemic metabolism by inducing free FA release through lipolysis and decreasing insulin secretion by pancreatic β-cells 48 . Similarly, IL-6 also modulates FA metabolism by inducing hepatic triglyceride secretion in response to infections and fasting 49 . ...
The interplay between the cardiovascular system, metabolism, and inflammation plays a central role in the pathophysiology of a wide spectrum of cardiovascular diseases, including heart failure. Here, we provide an overview of the fundamental aspects of the interrelation between inflammation and metabolism, ranging from the role of metabolism in immune cell function to the processes how inflammation modulates systemic and cardiac metabolism. Furthermore, we discuss how disruption of this immuno-metabolic interface is involved in the development and progression of cardiovascular disease, with a special focus on heart failure. Finally, we present new technologies and therapeutic approaches that have recently emerged and hold promise for the future of cardiovascular medicine.
... For example, acute exercise increases IL-6 100-fold without activating TNF or IL-1 beta, resulting in a constellation of biological changes generally considered anti-inflammatory 51 . Hyperthermia 52 and fasting 53 induce similar IL-6-increasing and health-promoting changes. Further, these studies generally support antidepressant or mood-enhancing properties as well, although these interventions only transiently increase IL-6. ...
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Because medical illness is associated with increased inflammation and an increased risk for treatment-resistant major depressive disorder, anti-cytokine therapy may represent a novel, and especially efficacious, treatment for depression. We hypothesized that blockade of the interleukin (IL)-6 signaling pathway with tocilizumab would decrease depression and related symptomatology in a longitudinal cohort of allogeneic hematopoietic stem cell transplantation (HCT) patients, a medically ill population with a significant inflammation and psychopathology. Patients undergoing allogeneic HCT received either a single dose of tocilizumab one day prior to HCT (n = 25), or HCT alone (n = 62). The primary outcome included depressive symptoms at 28 days post HCT; anxiety, fatigue, sleep, and pain were assessed at pretreatment baseline and days +28, +100, and +180 post HCT as secondary outcomes. Multivariate regression demonstrated that preemptive treatment with tocilizumab was associated with significantly higher depression scores at D28 vs. the comparison group (β = 5.74; 95% CI 0.75, 10.73; P = 0.03). Even after adjustment for baseline depressive symptoms, propensity score, and presence of acute graft-versus-host disease (grades II–IV) and other baseline covariates, the tocilizumab-exposed group continued to have significantly higher depression scores compared to the nonexposed group at D28 (β = 4.73; 95% CI 0.64, 8.81; P = 0.02). Despite evidence that IL-6 antagonism would be beneficial, blockade of the IL-6 receptor with tocilizumab among medically ill patients resulted in significantly more—not less—depressive symptoms.
... Another advantage of heat treatments is that hyperthermia treatments result in high levels of IL-6 without activating IL-1-beta or tumor necrosis factor (TNF) [53] potentially preventing cytokine storms (dysregulated immune response) because an increase of IL-6 by itself seems to decrease inflammation [54]. The isolated increase of IL-6 is the way exercise decreases inflammation [55], which is the same way for fasting [56] and for plant-rich diets [57]. The effect of hyperthermia on the coronavirus infection by way of the interferon function seems to be optimal at higher temperatures, 104.9 • F [58]. ...
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COVID-19 is a new contagious disease caused by a new coronavirus known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). COVID-19 is a disease that has reached every continent in the world; it has overloaded the medical system worldwide and it has been declared a pandemic by the World Health Organization. Currently there is no definite treatment for COVID-19. We realize that host immunity is a critical factor in the outcome of coronavirus 2 infection. Here, however, we review the pathophysiology of the disease with a focus on searching for what we can do to combat this new disease. From this, we find that coronavirus is sensitive to heat. We have thus focused on this area of vulnerability of the virus. The emphasis of this hypothesis is on the action of body heat—internal (fever) and external (heat treatment)—in activating the immune system and its antiviral activities, and specifically related to the coronavirus. We hypothesize from this review that heat treatments has the potential to prevent COVID-19 and to decrease the severity of mild and moderate cases of Coronavirus. We propose heat treatments for this uncontrolled worldwide coronavirus pandemic while studies are being done to test the effectiveness of heat treatments in the prevention and treatment of COVID-19.
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COVID-19 is a brand new contagious sickness caused by a brand new coronavirus referred to as intense acute breathing syndrome coronavirus 2 (SARS-CoV-2). COVID-19 is a disease that has reached each continent inside the global; it has overloaded the medical system international and it has been declared a plague by using the arena health agency. presently there are not any set up or tested treatments for COVID-19, that is permitted worldwide. Nanoparticles are described as stable colloidal particles ranging in size from 10 to 1000 nm. Nanoparticles provide many advantages to larger particles including multiplied surface-to-volume ratio and improved magnetic properties. Over the last few years, there was a regularly developing interest in the usage of nanoparticles in distinct biomedical packages inclusive of focused drug transport, hyperthermia, photoablation therapy, bioimaging and biosensors. in this review we've got hypothesize the class and synthesis of nanoparticles with diverse remedies along with photobiomodulation, drug shipping gadget, electrochemical nanotechnology biosensors, hydrothermotherapy and photocatalytic pastime which can be used for remedy and prevention of COVID-19 to lower the severity of moderate and slight instances of Coronavirus. We address current in addition to emerging therapies and prophylactic techniques that may allow us to efficaciously fight this pandemic and additionally can also assist to discover the key areas where nano-scientists can step in.
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Background Short-term markers of successful visceral adipose tissue (VAT) loss are needed. Urinary F2-isoprostanes may serve as a marker for intensified lipid metabolism, whereas circulating IL-6 may stimulate fat oxidation and enhance mobilization of VAT. Objective This pilot study was designed to explore the hypotheses that 1) reduction in VAT is associated with increase in IL-6, and 2) that increases in urinary F2-isoprostanes are associated with increases in IL-6 and reduction in VAT. Design Eighteen participants (age 60–75, BMI 30–40kg/m2) were randomized to either a very low carbohydrate diet (VLCD, <10:25: >65% energy from carbohydrate: protein: fat) or a low-fat diet (LFD) (55:25:20) for 8 weeks. Changes in fat distribution were assessed by magnetic resonance imaging (MRI). Four urinary F2-isoprostane isomers were quantified in 24-hour urine collection using LC-MS/MS analyses. Changes in four F2-isoprostane isomers were summarized using factor analysis (Δ-F2-isoprostane factor). Statistical significance was set at p < 0.1. Results Within the VLCD group, change in VAT was inversely associated with change in IL-6 (r = -0.778, p = 0.069) and change in F2-isoprostane factor (r = -0.690, p = 0.086), demonstrating that participants who maintained higher levels of F2-isoprostane factor across the intervention showed greater decreases in VAT. A positive relationship between change in F2-isoprostane factor and change in IL-6 was observed (r = 0.642, p = 0.062). In the LFD group, no significant associations between changes in VAT, F2-isoprostane factor, or IL-6 were observed. Conclusion Results from this exploratory study in older adults with obesity suggests that, in the context of a VLCD, IL-6 may be involved in VAT mobilization, and urinary F2-isoprostanes may reflect intensified oxidation of mobilized fatty acids. Trial Registration: NCT02760641. Registered 03 May 2016 – Retrospectively registered.
Obesity is an escalating global epidemic. As a direct result, millions of people will suffer from a multitude of health disorders, including type 2 diabetes, cancer and cardiovascular disease. The most effective and health-promoting methods of achieving and maintaining weight loss continue to be debated. Calorie restriction has long been used to promote weight loss and has recognised health benefits, but it can be hard to adhere to and has some health risks and side effects. Intermittent Fasting (IF) may be safer and easier to maintain and could trigger cellular pathways that promote cell survival over growth and reproduction. This may explain why intermittent fasting could improve health over and above the effects of weight loss. There are many different types of intermittent fasting patterns, with current studies focusing on ‘early time restricted feeding’ which uses knowledge of the circadian rhythm to enhance the effects of fasting. Health systems have yet to integrate IF as part of routine treatment protocols given the lack of long-term data on safety and efficacy. Patient awareness of IF is predominantly through self-research, health books and websites, which potentially raises safety concerns, particularly for patients on medications.
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IL-6 is an exercise-regulated myokine that has been suggested to increase lipolysis in fast twitch skeletal muscle. However, it is not known if a similar effect is present in slow twitch muscle. Furthermore, epinephrine increases IL-6 secretion from skeletal muscle suggesting that IL-6 could play a role in mediating the lipolytic effects of catecholamines. The purpose of this study was to determine if IL-6 stimulates skeletal muscle lipolysis in a fiber type dependent manner and is required for epinephrine-stimulated lipolysis in murine skeletal muscle. Soleus and EDL muscles from male C57BL/6J (WT) and IL-6(-/-) mice were incubated with 1µM (183 ng/ml) epinephrine or 75 ng/mL rIL-6 for 60 minutes. IL-6 treatment increased AMPK and STAT3 phosphorylation and glycerol release in isolated EDL but not soleus muscles from C57BL/6J mice. Conversely, epinephrine increased glycerol release in soleus but not EDL from C57BL/6J mice. Basal lipolysis was elevated in soleus muscle from IL-6(-/-) mice, and this was associated with increases in adipose triglyceride lipase (ATGL) and its co-activator CGI-58. The increase in ATGL content does not appear to be due to a loss of IL-6's direct effects as ex vivo treatment with IL-6 failed to alter the expression of ATGL mRNA in soleus. In summary, IL-6 stimulates lipolysis in glycolytic, but not oxidative muscle whereas the opposite fiber type effect is seen with epinephrine. The absence of IL-6 indirectly up-regulates lipolysis and this is associated with increases in ATGL and it co-activator CGI-58.
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The "portal hypothesis" proposes that the liver is directly exposed to free fatty acids and cytokines increasingly released from visceral fat tissue into the portal vein of obese subjects, thus rendering visceral fat accumulation particularly hazardous for the development of hepatic insulin resistance and type 2 diabetes. In the present study, we used a fat transplantation paradigm to (artificially) increase intra-abdominal fat mass to test the hypothesis that venous drainage of fat tissue determines its impact on glucose homeostasis. Epididymal fat pads of C57Bl6/J donor mice were transplanted into littermates, either to the parietal peritoneum (caval/systemic venous drainage) or, by using a novel approach, to the mesenterium, which confers portal venous drainage. Only mice receiving the portal drained fat transplant developed impaired glucose tolerance and hepatic insulin resistance. mRNA expression of proinflammatory cytokines was increased in both portally and systemically transplanted fat pads. However, portal vein (but not systemic) plasma levels of interleukin (IL)-6 were elevated only in mice receiving a portal fat transplant. Intriguingly, mice receiving portal drained transplants from IL-6 knockout mice showed normal glucose tolerance. These results demonstrate that the metabolic fate of intra-abdominal fat tissue transplantation is determined by the delivery of inflammatory cytokines to the liver specifically via the portal system, providing direct evidence in support of the portal hypothesis.
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The contribution of interleukin (IL)-6 signaling in obesity-induced inflammation remains controversial. To specifically define the role of hepatic IL-6 signaling in insulin action and resistance, we have generated mice with hepatocyte-specific IL-6 receptor (IL-6R) a deficiency (IL-6RaL-KO mice). These animals showed no alterations in body weight and fat content but exhibited a reduction in insulin sensitivity and glucose tolerance. Impaired glucose metabolism originated from attenuated insulin-stimulated glucose transport in skeletal muscle and fat. Surprisingly, hepatic IL-6Ra-disruption caused an exaggerated inflammatory response during euglycemic hyperinsulinemic clamp analysis, as revealed by increased expression of IL-6, TNF-a, and IL-10, as well as enhanced activation of inflammatory signaling such as phosphorylation of IkBa. Neutralization of TNF-a or ablation of Kupffer cells restored glucose tolerance in IL-6RaL-KO mice. Thus, our results reveal an unexpected role for hepatic IL-6 signaling to limit hepatic inflammation and to protect from local and systemic insulin resistance.
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Adipose tissue inflammation is linked to the pathogenesis of insulin resistance. In addition to exerting death-promoting effects, the death receptor Fas (also known as CD95) can activate inflammatory pathways in several cell lines and tissues, although little is known about the metabolic consequence of Fas activation in adipose tissue. We therefore sought to investigate the contribution of Fas in adipocytes to obesity-associated metabolic dysregulation. Fas expression was markedly increased in the adipocytes of common genetic and diet-induced mouse models of obesity and insulin resistance, as well as in the adipose tissue of obese and type 2 diabetic patients. Mice with Fas deficiency either in all cells or specifically in adipocytes (the latter are referred to herein as AFasKO mice) were protected from deterioration of glucose homeostasis induced by high-fat diet (HFD). Adipocytes in AFasKO mice were more insulin sensitive than those in wild-type mice, and mRNA levels of proinflammatory factors were reduced in white adipose tissue. Moreover, AFasKO mice were protected against hepatic steatosis and were more insulin sensitive, both at the whole-body level and in the liver. Thus, Fas in adipocytes contributes to adipose tissue inflammation, hepatic steatosis, and insulin resistance induced by obesity and may constitute a potential therapeutic target for the treatment of insulin resistance and type 2 diabetes.
Use of the real-time polymerase chain reaction (PCR) to amplify cDNA products reverse transcribed from mRNA is on the way to becoming a routine tool in molecular biology to study low abundance gene expression. Real-time PCR is easy to perform, provides the necessary accuracy and produces reliable as well as rapid quantification results. But accurate quantification of nucleic acids requires a reproducible methodology and an adequate mathematical model for data analysis. This study enters into the particular topics of the relative quantification in real-time RT-PCR of a target gene transcript in comparison to a reference gene transcript. Therefore, a new mathematical model is presented. The relative expression ratio is calculated only from the real-time PCR efficiencies and the crossing point deviation of an unknown sample versus a control. This model needs no calibration curve. Control levels were included in the model to standardise each reaction run with respect to RNA integrity, sample loading and inter-PCR variations. High accuracy and reproducibility (<2.5% variation) were reached in LightCycler PCR using the established mathematical model.
The ability to store energy in the form of energy-dense triacylglycerol and to mobilize these stores rapidly during periods of low carbohydrate availability or throughout the strong metabolic demand is a highly conserved process, absolutely essential for survival. In the industrialized world the regulation of this pathway is viewed as an important therapeutic target for disease prevention. Adipose tissue lipolysis is a catabolic process leading to the breakdown of triacylglycerols stored in fat cells, and release of fatty acids and glycerol. Mobilization of adipose tissue fat is mediated by the MGL, HSL and ATGL, similarly functioning enzymes. ATGL initiates lipolysis followed by the actions of HSL on diacylglycerol, and MGL on monoacylglycerol. HSL is regulated by reversible phosphorylation on five critical residues. Phosphorylation alone, however, is not enough to activate HSL. Probably, conformational alterations and a translocation from the cytoplasm to lipid droplets are also involved. In accordance, Perilipin functions as a master regulator of lipolysis, protecting or exposing the triacylglycerol core of a lipid droplet to lipases. The prototype processes of hormonal lipolytic control are the β-adrenergic stimulation and suppression by insulin, both of which affect cytoplasmic cyclic AMP levels. Lipolysis in adipocytes is an important process in the management of body energy reserves. Its deregulation may contribute to the symptoms of type 2 diabetes mellitus and other pathological situations. We, herein, discuss the metabolic regulation and function of lipases mediating mammalian lipolysis with a focus on HSL, quoting newly identified members of the lipolytic proteome.
The chemokine CXC ligand-1 (CXCL-1) is a small cytokine that elicits effects by signalling through the chemokine receptor CXCR2. CXCL-1 has neutrophil chemoattractant activity, is involved in the processes of angiogenesis, inflammation and wound healing, and may possess neuroprotective effects. The aim of this study was to unravel the mechanisms whereby CXCL-1 is regulated by exercise inmice. After a single bout of exercise, CXCL-1 protein increased in serum(2.4-fold), and CXCL-1 mRNA in muscle (6.5-fold) and liver (41-fold). These increases in CXCL-1 were preceded by increases in serum interleukin-6 (IL-6) and muscle IL-6 mRNA. In contrast, exercise-induced regulation of liver CXCL-1 mRNA expression was completely blunted in IL-6 knockout mice. Based on these findings, we examined the possible existence of a muscle-to-liver axis by overexpressing IL-6 in muscles. This resulted in increases in serum CXCL-1 (5-fold) and liver CXCL-1 mRNA expression (24-fold) compared with control. Because IL-6 expression and release are known to be augmented during exercise in glycogen-depleted animals, CXCL-1 and IL-6 expression were examined after exercise in overnight-fasted mice.We found that fasting significantly augmented serum CXCL-1, and CXCL-1 expression in liver and muscle. Taken together, these data indicate that liver is the main source of serum CXCL-1 during exercise in mice, and that the CXCL-1 expression in the liver is regulated by muscle-derived IL-6.
Obesity represents a low-grade inflammatory disease and appears a risk factor for insulin resistance, but little is known on whether this may contribute to the development of autoimmune inflammatory diseases. The aim of this work was to study the early-life diet-induced obesity in Lewis rats which are known to be highly susceptible to autoimmunity. Obesity was induced by reduced litter size (4 pups per litter) followed by high-fat diet (SHF rats). Control rats (8 pups per litter) were fed with standard diet (CN rats). Oral glucose tolerance test (3 g glucose per kg b.w.) was performed by intra-gastric tube in conscious rats after 12 h fast. Adipocyte size was assessed by light microscope after collagenase digestion. Hypothalamic arcuate (ARC) and paraventricular nuclei (PVN) were isolated by the punching technique. Target mRNAs were quantified by real-time PCR with the use of TaqMan probes and primers. Serum hormones (leptin, ghrelin, adiponectin, visfatin and insulin) were assayed by specific RIAs . During the experimental period SHF rats had the same body weight gain and caloric intake as CN rats. At the age of 8 weeks SHF rats showed increased epididymal fat mass and adipocyte volume, impaired glucose tolerance, normal basal fasting insulin, visfatin, and ghrelin level, but decreased adiponectin and high leptin level. In the ARC, the SHF rats showed increased expression of mRNA for orexigenic neuropeptide Y (NPY), agouti-related protein (AgRP) and anorexigenic pro-inflammatory cytokine IL-6. In the PVN, the SHF rats showed increased expression of mRNA for anorexigenic melanocortin 4 receptor (MC4R) and IL-6. Overexpression of orexigenic NPY and AgRP in the ARC indicates leptin resistance in SHF rats. The increased expression of MC4R in PVN points to the activation of melanocortin anorexigenic system which, along with increased hypothalamic IL-6, might prevent the animals from overfeeding. Higher adiposity in these rats results from the high fat-diet composition and not from increased caloric intake. Furthermore, enhanced leptin production appears the main factor indicating the predisposition to autoimmunity in these overfed rats.
Our previous studies have demonstrated that activation of alpha(1)-adrenergic receptors (ARs) increased interleukin-6 (IL-6) mRNA expression and protein secretion, which is probably an important yet unknown mechanism contributing to the regulation of cardiac function. Using Rat-1 fibroblasts stably transfected with the alpha(1A)-AR subtype and primary mouse neonatal cardiomyocytes, we elucidated the basic molecular mechanisms responsible for the alpha(1)-AR modulation of IL-6 expression. IL-6 mRNA production mediated by alpha(1)-AR peaked at 1 to 2 h. Studies of the mRNA decay rate indicated that alpha(1)-AR activation enhanced IL-6 mRNA stability. Analysis of IL-6 promoter activity using a series of deletion constructs indicated that alpha(1)-ARs enhanced IL-6 transcription through several transcriptional elements, including nuclear factor kappaB (NF-kappaB). Inhibition of alpha(1)-AR mediated IL-6 production and secretion by actinomycin D and the increase of intracellular IL-6 levels by alpha(1)-AR activation suggest that alpha(1)-AR mediated IL-6 secretion through de novo synthesis. Both IL-6 transcription and protein secretion mediated by alpha(1)-ARs were significantly reduced by chemical inhibitors for p38 mitogen-activated protein kinase (MAPK) and NF-kappaB and by a dominant-negative construct of p38 MAPK. Serum level of IL-6 was elevated in transgenic mice expressing a constitutively active mutant of the alpha(1A)-AR subtype but not in a similar mouse model expressing the alpha(1B)-AR subtype. Our results indicate that alpha(1)-ARs stimulated IL-6 expression and secretion through regulating both mRNA transcription and stability, involving p38 MAPK and NF-kappaB pathways.