adipocytes Exercise-inducible factors to activate lipolysis in

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Exercise-inducible factors to activate lipolysis in
adipocytes
Takeshi Hashimoto, Koji Sato and Motoyuki Iemitsu
J Appl Physiol 115:260-267, 2013. First published 16 May 2013; doi:10.1152/japplphysiol.00427.2013
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J Appl Physiol 115: 260–267, 2013.
First published May 16, 2013; doi:10.1152/japplphysiol.00427.2013.
Exercise-inducible factors to activate lipolysis in adipocytes
Takeshi Hashimoto, Koji Sato, and Motoyuki Iemitsu
Faculty Sport & Health Science, Ritsumeikan University, Shiga, Japan
Submitted 4 April 2013; accepted in final form 9 May 2013
obesity; lipid droplet; lipid droplet–associated protein; mitochondria
in adipocytes is a central feature
of obesity and metabolic syndrome. Excess energy is primarily
stored as triacylglycerol (TAG) in the lipid droplets (LDs) of
mammalian adipose tissue, and those TAG reserves are hydrolyzed to supply fatty acids (FAs) to various tissues by a process
called lipolysis, when the stored energy is required such as
during starvation and exercise. Thus, physiological strategies
such as exercise training aimed toward fat loss by active
lipolysis in adipocytes (i.e., fat mobilization) and FA oxidation
in muscles (i.e., fat utilization) have become preferred therapeutic agents against metabolic disorders. However, remarkably little is known about molecular mechanisms by which
exercise training augments lipid mobilization.
By interacting with one another, LD-associated proteins
such as perilipin, hormone sensitive lipase (HSL), adipose
triglyceride lipase (ATGL), and its coactivator comparative
gene identification (CGI)-58 [also called ␣,␤-hydrolase domain-containing (ABHD) 5] are purported to play important
roles in regulating fat storage and mobilization (1, 13, 14, 17,
43, 45). In addition to elaborate interactions among LDassociated proteins, it is possible that the mitochondria may
play a pivotal role, not only in determining the oxidative
EXCESSIVE LIPID ACCUMULATION
Address for reprint requests and other correspondence: T. Hashimoto
(e-mail: thashimo@fc.ritsumei.ac.jp).
260
capacity, but also the protein kinase A (PKA)-mediated lipolysis in adipose tissue, thereby regulating adipocyte lipid metabolism. We assume that mitochondrial oxidative capacity
affects PKA-mediated lipolytic activity in adipose tissue, likely
due to the mechanism that enhanced mitochondrial oxidation
within adipocytes may be required to provide energy and cyclic
AMP for PKA-mediated lipolysis and other adipocyte metabolic functions during exercise (8). This assumption was supported by the observation of reduced lipolysis upon deficient
mitochondrial respiration in isolated human fat cells following
treatment with oxidative phosphorylation inhibitors antimycin
A and 2,4-dinitrophenol (12). Actually, in rodent models,
mitochondrial content is reduced in adipose tissue of animals
with insulin resistance and type 2 diabetes mellitus (T2DM) (4,
40). Similarly, a decrease in mitochondrial ATP production
results in the inhibition of both FA synthesis and lipolytic
action of catecholamines (6, 8). Inversely, augmented mitochondrial oxidative activity would be beneficial for covering
the energy cost of the activation of lipolysis in white adipocytes (8).
Although few reports have examined the effects of exercise
training on the regulation of LD-associated proteins or mitochondrial biogenesis and mitochondrial function in adipocytes,
Wohlers et al. demonstrated that acute exercise elevated the
expression of ATGL and HSL Ser660 phosphorylation in
adipose tissue (41). Additionally, Ogasawara et al. demonstrated that endurance exercise training elevated protein expression of ATGL in adipocytes (27). The study also demonstrated that exercise training increased mRNA expression of
peroxisome proliferator-activated receptor (PPAR)-gamma coactivator 1alpha (PGC-1␣), a master regulator of mitochondrial
biogenesis, as well as mitochondrial proteins in rat adipose
tissue. These previous results indicated that exercise-induced
increase in circulating catecholamine levels may be one potential mechanism of augmented mitochondrial biogenesis (36).
In contrast to adipose tissue, many studies have focused on
skeletal muscle mitochondrial biogenesis. To date, it has been
reported that exercise increases the expression of PGC-1␣
mainly through two pathways: AMP-activated protein kinase
(AMPK) and calcium signaling pathways (3, 28, 38). McConell et al.
proposed that N1-(␤-D-ribofuranosyl)-5-aminoimidazole-4-carboxamide (AICAR) and caffeine, the activators of AMPK and
cellular calcium levels, respectively, increase mitochondrial
biogenesis, in part via interactions with nitric oxide synthase
(NOS)/nitric oxide (NO) (23). Additionally, we demonstrated
that metabolic signals induced by lactate exposure can stimulate the transcription of genes involved in mitochondrial biogenesis in L6 myotubes, and reactive oxygen species (ROS),
especially hydrogen peroxide (H2O2), were involved in the
regulation of gene expression, including PGC-1␣ (16). Consistent with this notion, the contraction-induced increase in
gene expression of PGC-1␣, mitochondrial uncoupling protein
3 (UCP3) and hexokinase II (HKII) in primary rat skeletal
8750-7587/13 Copyright © 2013 the American Physiological Society
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Hashimoto T, Sato K, Iemitsu M. Exercise-inducible factors to
activate lipolysis in adipocytes. J Appl Physiol 115: 260 –267, 2013. First
published May 16, 2013; doi:10.1152/japplphysiol.00427.2013.—We
examined the effects of exercise training on the levels of lipid droplet
(LD)-associated and mitochondria-related proteins in diet-induced obese
(DIO) rats. Furthermore, we assessed putative factors induced by exercise
to activate lipolysis in differentiated 3T3-L1 adipocytes. DIO Wistar
male rats (age 20 wk) were divided into sedentary control (SED, n ⫽ 7)
and exercise training (EX, n ⫽ 7) groups. EX animals were subjected to
treadmill running (25 m/min, 1 h/day, 5 days/wk) for 6 wk. Epididymal
fat was dissected and used for protein analyses. 3T3-L1 adipocytes were
incubated with media containing hydrogen peroxide (H2O2), sodiumlactate, caffeine, AICAR, or SNAP (NO donor) for 6 h, or 1 mM H2O2
for 15 min, followed by incubation with normal media for up to 24 h
total. Protein expression levels and lipolytic activities were biochemically
assayed. Epididymal fat significantly decreased in EX animals compared
with SED animals. Levels of cytochrome c oxidase (COx), perilipin,
hormone sensitive lipase (HSL), and adipose triglyceride lipase (ATGL)
proteins in epididymal fat pads of EX animals were significantly increased compared with those in SED animals. In 3T3-L1 cells, glycerol
or fatty acid release was significantly increased by all treatments. Lactate
or SNAP significantly increased PGC-1␣ expression, and H2O2 significantly increased COx protein levels compared with controls. Expression
of perilipin, HSL, ATGL, or comparative gene identification (CGI)-58
was significantly increased by all treatments. By increasing lipolytic
activity in adipocytes, the exercise-inducible factors are attractive therapeutic effectors against LD-associated metabolic diseases.
Active Lipolysis in Adipocytes
MATERIALS AND METHODS
Animal care. Ethical approval for this study was obtained from the
Committee on Animal Care at the University of Tsukuba and Ritsumeikan University. Male Wistar rats (220 –250 g, 10 wk old) were
obtained (Charles River Japan, Kanagawa, Japan) and cared for
according to the guiding principles for the care and use of animals
based on the Declaration of Helsinki. The rats were housed individually in an animal facility under controlled conditions (12:12-h
light-dark cycle). To induce obesity and hyperglycemia, the rats were
allowed food/water ad libitum and placed on a purified high-sucrose
diet (68% of kcal from sucrose, 20% from protein, and 12% from fat)
for 14 wk according to our previous studies (31, 32). After 14 wk, the
obese animals were randomly assigned to two groups: sedentary
control (SED, n ⫽ 7) and exercise training (EX, n ⫽ 7). All animals
continued the high-sucrose diet during the 6-wk experimental period.
Exercise training protocol. To orient and accustom animals to
treadmill exercise, the exercise-training group was initially run on a
rodent treadmill at 10 –15 m/min for 3 days. Subsequently, rats were
run on the treadmill for 1 h at 25 m/min without incline 5 days/wk for
6 wk. Exercise intensity was kept constant during the training period.
Seventy-two hours after the final bout of exercise training, epididymal
fat was quickly removed, weighed, rinsed in ice-cold saline, and
frozen in liquid nitrogen.
Acute exercise protocol. Male Wistar rats (220 –250 g, 10 wk old)
were divided into sedentary control (CON, n ⫽ 6) and acute exercise
(AEX, n ⫽ 6) groups. Prior to the acute exercise, rats were accustomed to the treadmill at a speed of 30 m/min for 30 min. One week
Hashimoto T et al.
261
after the acclimation, the rats of the AEX group ran on the treadmill
for 30 min at 30 m/min without incline. Immediately after exercise,
epididymal fat was quickly removed, weighed, rinsed in ice-cold
saline, and frozen in liquid nitrogen.
Cell culture protocol. All reagents for cell culture were obtained
from Wako (Osaka, Japan) unless otherwise mentioned. The cell
culture procedure was the same as that described in our previous
report (17). Briefly, 3T3-L1 cells were maintained in DMEM/10%
fetal bovine serum (FBS). For differentiation, confluent cells (day 0)
were treated with differentiation media, which is a hormone mixture
containing 1 ␮M dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), and 5 ␮g/ml insulin in DMEM/10% FBS. After 48 h
(day 2), the differentiation media was removed and cells were further
cultured in growth media (DMEM/10% FBS supplemented with 5
␮g/ml insulin). The growth media was replaced with fresh growth
media every 3 days up to the experimental days.
Differentiated 3T3-L1 adipocytes were incubated with media containing 10 ␮M H2O2, 10 mM sodium-lactate, 5 mM caffeine, 2 mM
AICAR (Sigma), 100 ␮M SNAP for 6 h, or 1 mM H2O2 for 15 min,
followed by incubation with normal fresh media for 18 h (23 h and 45
min for 1 mM H2O2). After 24 h from the beginning of the incubation
with H2O2, sodium-lactate, caffeine, AICAR, or SNAP cells were
used for assays of lipolytic activity or Western blotting.
Lipolytic stimulation. When lipolytic activity was analyzed, 2%
FA-free BSA (Wako) was used instead of 10% FBS. Lipolytic
stimulation was added to differentiated 3T3-L1 adipocytes as follows:
after washing twice with Hank’s buffer, cells were incubated with
DMEM containing 10% FBS/4mM L-glutamine (Gibco)/25 mM
HEPES (pH 7.4)/0.5 mM IBMX/10 ␮M isoproterenol at 37°C.
Western blotting. Tissue samples (300 mg each) were homogenized
in 2 ml of buffer containing 20 mM Tricine (pH 7.8), 250 mM
sucrose, 10 mM NaF, 1 mM PMSF, a protease inhibitor mixture
(Sigma-Aldrich, St. Louis, MO), and phosphatase inhibitor cocktail
(Sigma-Aldrich), and incubated on ice for 20 min. Tissues were then
homogenized using a Potter-Elvehjem tissue homogenizer (AS ONE,
Osaka, Japan) on ice with 10 gentle strokes with the motor-driven
pestle at 2,500 rpm. Homogenates were mixed with four times the
sample volume of cold acetone (⫺20°C) and incubated overnight at
⫺20°C. The samples were then centrifuged at 9,000 g for 15 min at
4°C and the supernatants were disposed. Acetone was evaporated and
the pellets were resuspended with buffer containing 20 mM Tris (pH
7.4), 1 mM EDTA, and 0.1% Triton X-100, and protein concentration
was measured. The protein extracts were subjected to SDS-PAGE and
transferred to a nitrocellulose membrane.
3T3-L1 cells were washed with PBS and directly dissolved in the
heated SDS-PAGE sample buffer. Aliquots of the extracts were
subjected to SDS-PAGE and transferred to a nitrocellulose membrane.
Proteins were probed with an antibody to CGI-58 (sc-102285; Santa
Cruz Biotechnology), HSL (#4107; Cell Signaling), ATGL (#2138;
Cell Signaling), perilipin (GPEE; Progen, Dara, Australia), PGC-1␣
(ST-1202; Calbiochem), electron transport chain protein cytochrome
c oxidase (COx) (ab54575; abcam), endothelial NOS (eNOS)
(#610297; Transduction Laboratories), phospho-NOS (p-NOS) (sc12972; Santa Cruz Biotechnology), AMPK (#2793; Cell Signaling),
phospho-AMPK (p-AMPK) (#2531; Cell Signaling), calcium/calmodulin-dependent protein kinase II (CaMKII) (#3362; Cell Signaling), phospho-CaMKII (p-CaMKII) (#3361; Cell Signaling) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (G9545; Sigma-Aldrich)
detected by the enhanced chemiluminescence method (Amersham Biosciences). The immunoblotted membranes were scanned with an ImageQuant LAS-4000 (GE Healthcare) luminescent image analyzer and
the optical density of each specific band was analyzed with ImageQuant
TL software (GE Healthcare). Densitometric analysis of immunoblots
was normalized to GAPDH.
Glycerol release, FA release, and triglyceride measurements.
3T3-L1 cells were grown in 12-well dishes. Differentiated cells were
pretreated with either 10 ␮M H2O2, 10 mM sodium-lactate, 5 mM
J Appl Physiol • doi:10.1152/japplphysiol.00427.2013 • www.jappl.org
muscle cells is dependent on ROS production (34). Similarly,
it was demonstrated in a mouse fibroblastic cell line that H2O2
treatment increased PGC-1␣ through increased cAMP response element-binding protein (CREB), the important transcription factor for PGC-1␣ gene expression (3, 35). Furthermore, in mouse skeletal muscle, Koves et al. demonstrated that
either acute exercise or PGC-1␣ overexpression stimulated
gene expression of HSL, ATGL, CGI-58, and perilipin 5.
Perilipin 5 belongs to a family of lipid coat protein enriched in
oxidative muscles, suggesting that there is an intimate molecular connection between LD regulation and mitochondrial
metabolism (20). As a whole, these findings led us to suspect
that these exercise-inducible factors, known to be generated in
skeletal muscle, could travel from muscle to adipose tissue
and/or could be generated in adipose tissue, thereby increasing
lipolytic activity in adipocytes by means of increased mitochondrial biogenesis and LD-associated proteins. This idea
was tested in the investigation reported here.
Here we examined the effects of exercise training on levels
of LD-associated proteins and mitochondrial biogenic signaling in adipose tissue of exercised diet-induced obese (DIO)
rats, in which enhanced lipolytic activity would be more
appreciated than in nonobese rats. We also examined the
effects of compounds that are known to reproduce some of the
exercise effects in skeletal muscle such as H2O2, sodiumlactate, caffeine, AICAR, and the NO donor S-nitroso-Nacetyl-penicillamine (SNAP), on the expression of LD-associated proteins and mitochondrial biogenic signaling to explore
putative factors induced by exercise training to activate lipolysis in differentiated 3T3-L1 adipocytes. We hypothesized that
exercise training would increase the levels of LD-associated
proteins and mitochondrial biogenic signaling in DIO rats.
Furthermore, we hypothesized that ROS, lactate-inducible signals, increased intracellular Ca2⫹ levels, AMPK, and NO
would increase lipolytic activity in adipocytes.
•
Table 1. Effect of exercise training on body weight and
epididymal fat weight
Body weight, g
Epididymal fat weight, g
SED
EX
696.2 ⫾ 9.7
26.2 ⫾ 1.7
613.9 ⫾ 21.9*
21.1 ⫾ 2.3*
Values are means ⫾ SEM. *P ⬍ 0.05 vs. SED group.
caffeine, 2 mM AICAR, 100 ␮M SNAP for 6 h, or 1 mM H2O2 for
15 min, and then washed twice with Hank’s buffer and incubated with
DMEM containing 2% FA-free BSA (Sigma)/20 mM HEPES (pH
7.4) with or without 0.5 mM IBMX and 10 ␮M isoproterenol at 37°C.
After incubation for 3 h, the media was collected and assayed for
glycerol and FA content using the triglyceride E-test kit and NEFA
C-test kit, respectively (Wako).
Statistical analysis. Statistical analyses were performed by unpaired t-tests or one-way ANOVA, as appropriate. Bonferroni post
hoc test was used as needed, and significance was set at P ⬍ 0.05. All
results are presented as means ⫾ SEM.
RESULTS
Hashimoto T et al.
cle are also inducible in adipose tissue in response to a single bout
of exercise. For this purpose, we assessed the p-NOS/eNOS ratio,
p-AMPK/total AMPK ratio, and p-CaMKII/total CaMKII ratio,
which would reflect NO production, AMPK activity, and intracellular Ca2⫹ concentration, respectively, in adipose tissue dissected immediately after exercise. As shown in Fig. 3, acute
exercise significantly increased p-NOS/eNOS and p-AMPK/total
AMPK ratios, suggesting that NO production and AMPK activity
may be increased by a single bout of exercise in adipose tissue.
Effects of putative exercise-inducible factors on lipolytic
activity. In contrast to NO and AMPK, which could be generated and activated, respectively, in adipocyte tissue in response
to acute exercise (Fig. 3), lactate produced in skeletal muscle
during exercise circulates through the body, including to adipose tissue (15). Furthermore, adipose tissue also produces
lactate (18), and in skeletal muscle cells lactate activates H2O2
production (16). Thus, we posited that lactate and H2O2 are
also exercise-inducible factors in adipose tissue during exercise. Although the p-CaMKII/total CaMKII ratio was not
altered in response to acute exercise, we attempted to assess the
effect of increased intracellular calcium levels. Hence, we
determined whether those exercise-inducible factors could activate lipolysis in 3T3-L1 adipocytes. Glycerol release and FA
release into the media during lipolytic stimulation were measured for cells treated with either 10 ␮M or 1 mM H2O2, 10
mM sodium-lactate, 5 mM caffeine, 2 mM AICAR, or 100 ␮M
SNAP. Treatments with sodium-lactate, caffeine, or SNAP
each significantly increased glycerol release (Fig. 4), which
represents total lipolytic activity in adipocytes (because glycerol produced during lipolysis is not efficiently phosphorylated
and used for TAG synthesis due to the poor activity of glycerol
kinase in adipocytes) (21, 29). On the other hand, 1 mM H2O2,
caffeine, and AICAR each significantly increased FA release
(Fig. 4).
Effects of putative exercise-inducible factors on mitochondrial biogenic signaling. We next examined whether exerciseinducible factors could increase mitochondrial biogenic signaling in 3T3-L1 adipocytes. Because 10 ␮M H2O2 failed to have
an effect on lipolytic activity (Fig. 4), we examined the effect
of 1 mM H2O2 for the H2O2 treatment. Sodium-lactate (P ⬍
0.01) and SNAP (P ⬍ 0.05) each significantly increased
PGC-1␣ expression, and 1 mM H2O2 significantly increased
COx expression (P ⬍ 0.05) (Fig. 5). H2O2, caffeine, or AICAR
did not increase PGC-1␣ expression (P ⫽ 0.08, 0.06, and 0.18,
respectively); and sodium-lactate, caffeine, AICAR, and SNAP
did not increase COx expression (P ⫽ 0.10, 0.24, 0.21, and
0.08, respectively).
Fig. 1. Expression of mitochondria-related proteins such as PGC-1␣ and COx were examined
in epididymal fat in sedentary control (SED,
n ⫽ 7) and exercise training (EX, n ⫽ 7)
groups. PGC-1␣ expression in EX was increased more than twofold compared with that
in SED. Expression of COx in EX was significantly increased compared with that in SED.
*P ⬍ 0.05 vs. SED group.
Protein content
(Arbitrary Units)
PGC1
COx
4
3
2
1
0
SED
EX
3
*
2
1
0
SED
EX
Effect of exercise training on body weight, epididymal fat
weight, mitochondrial biogenic signaling, and LD-associated
protein levels. As expected, exercise reduced body weights and
epididymal fat pad weights, thereby confirming a physiological
effect of exercise (Table 1).
Given that inhibition of mitochondrial respiration by antimycin A and 2,4-dinitrophenol decreased lipolysis in adipocytes (12), we hypothesized that mitochondrial oxidative
activity is crucial for active lipolysis in adipose tissue, thereby
providing a potential mechanism for an exercise trainingmediated decrease in fat pad weight. Thus, we determined
levels of mitochondrial biogenic signaling in epididymal fat for
SED and EX rats. PGC-1␣, a master regulator of mitochondrial
biogenesis, did not increase (P ⫽ 0.1), but COx significantly
increased in response to endurance training (Fig. 1).
We also examined expression of LD-associated proteins in
epididymal fat in response to exercise training. The EX group
showed significantly higher expression of HSL, perilipin, and
ATGL in epididymal fat than in the SED group (P ⬍ 0.05).
Expression of CGI-58 in the EX group tended to increase
compared with the SED group (P ⫽ 0.05) (Fig. 2).
Effects of acute exercise on calcium signals, NO, and AMPK
responses in epididymal fat. We explored exercise-inducible
factors in adipose tissue that may potentially elicit augmented
mitochondrial biogenic signaling and LD-associated protein expression by exercise training. Therefore, we examined whether
exercise-inducible factors known to be generated in skeletal mus-
•
Protein content
(Arbitrary Units)
262
*
2
1
SED
EX
3
P=0.05
2
1
0
SED
EX
ATGL
*
3
2
1
SED
EX
4
*
Fig. 2. Expression of lipid droplet (LD)-associated proteins such as perilipin, CGI-58,
HSL, and ATGL were examined in epididymal fat in sedentary control (SED, n ⫽ 7) and
exercise training (EX, n ⫽ 7) groups. Expression of HSL, perilipin, and ATGL in epididymal fat was significantly higher in the EX
group than the SED group. Expression of
CGI-58 in the EX group tended to increase
compared with that of the SED group (P ⫽
0.05). *P ⬍ 0.05 vs. SED group.
3
2
1
0
Effects of putative exercise-inducible factors on LD-associated proteins. We examined whether exercise-inducible factors
could increase the expression of LD-associated proteins in
3T3-L1 adipocytes. H2O2, sodium-lactate, caffeine, and SNAP
each significantly increased HSL expression; and H2O2, sodiumlactate, caffeine, and AICAR each significantly increased ATGL
expression (Fig. 6). Furthermore, sodium-lactate and SNAP each
significantly increased perilipin expression, whereas H2O2, sodium-lactate, caffeine, AICAR, and SNAP each significantly increased CGI-58 expression (Fig. 6).
DISCUSSION
The primary novel findings of the present study are that
prolonged endurance exercise training increased the expression
of LD-associated proteins and mitochondrial biogenic signaling
in adipose tissue of DIO rats. Furthermore, exercise-inducible
factors such as lactate, ROS, calcium signals, AMPK, and NO,
some of which could be generated in adipose tissue, and some of
which could be transported from skeletal muscle, were able to
enhance lipolytic activity in differentiated 3T3-L1 adipocytes.
Previous studies have demonstrated that exercise training
enhanced lipolytic activity in adipose tissues in rats (7, 27),
hamsters (2), overweight men (5), obese postmenopausal
women (44), or obese women with polycystic ovary syndrome
(25), and several potential mechanisms underlying the augmentation of adipocyte lipolysis during exercise have been
proposed. For example, Campbell et al. suggested that exercise
training enhances lipolytic activity through the augmented
activity of glucocorticoids concomitant with increased levels of
two major lipolytic enzymes, HSL and ATGL, in adipose
tissue (2). Ogasawara et al. suggested that exercise training
enhances lipolytic activity through elevated levels of ATGL in
adipocytes, concomitant with lower levels of plasma insulin
(27). Furthermore, Sutherland et al. suggested that exercise
SED
EX
training increased mitochondrial biogenesis in rat adipose tissue through an increase in circulating catecholamine levels
(36). In the present study, we found that exercise training
significantly increased, or tended to increase, levels of perilipin
or CGI-58, respectively, in addition to increased HSL and
ATGL in epididymal fat tissue. Perilipin and CGI-58 are
crucial for the activity of the major lipolytic enzymes HSL and
ATGL in adipocytes. Depending on the phosphorylation status,
perilipin blocks HSL in basal condition or recruits HSL to LD
surfaces in response to PKA stimulation, and reversibly docks
CGI-58, an ATGL activator, and the resulting ATGL/CGI-58
complex efficiently hydrolyzes TAG to diacylglycerol and FA
(17). Actually, perilipin expression in adipocytes is decreased
with obesity in humans (26). Furthermore, perilipin-null mice
showed elevated basal lipolysis but decreased catecholaminestimulated lipolysis, and increased insulin resistance (37). On
the other hand, perilipin overexpressed in mice protected
against DIO (24). Taken together, the increased expression
levels of LD-associated proteins in epididymal fat may be one
potential mechanism for the exercise-induced weight loss in
DIO rats in the present study, through the augmented capacity
of lipolysis.
We also examined levels of mitochondrial biogenic signaling in white adipose tissue in response to exercise training.
Although it was not statistically significant, expression of
PGC-1␣ in the exercise training group was more than twice
that in the SED control group. Furthermore, COx protein
expression was significantly increased by exercise training. As
mentioned below, we explored putative factors induced by
exercise training to activate mitochondrial biogenic signaling
and found that lactate, ROS, and NO increased the levels of
mitochondrial biogenic signaling. Thus, lactate, ROS, and NO
might be potential mechanisms for augmented mitochondrial
biogenic signaling in white adipose tissue in addition to circu-
Protein content
(Arbitrary Units)
HSL
0
Protein content
(Arbitrary Units)
3
0
263
Hashimoto T et al.
CGI-58
Protein content
(Arbitrary Units)
Protein content
(Arbitrary Units)
Perilipin
•
264
p-NOS/eNOS
p-NOS
Protein content
(Arbitrary Units)
eNOS
*
2
1.5
1
0.5
0
CON
p-AMPK
t-AMPK
4
*
3
2
1
0
CON
p-CaMK/
total CaMK
AEX
p-CaMK
t-CaMK
Hashimoto T et al.
in (8)]. We consider elevated levels of mitochondrial biogenic
signaling, and hence, elevated oxidative activity in white adipose tissue to be supportive of major metabolic pathways
including lipolysis. Another limitation of the study is that we
solely analyzed epididymal adipose tissue. However, as noted
previously [see (8)], there are known metabolic differences
between fat pads (33, 39). The extent to which these metabolic
responses to exercise-inducible factors are achieved in fat pads
other than epididymal fat warrants further study.
Next, we examined whether putative exercise-inducible factors known to be generated in skeletal muscle also could be
increased in adipose tissue. Although we could not test whether
exercise-inducible factors traveled from muscle to adipose
tissue or were generated in adipose tissue, we found that NO
production and AMPK activity may be increased by a single
bout of exercise in adipose tissue. This interpretation is supported by a previous study demonstrating that AMPK was
activated during lipolysis in adipocytes due to an increase in
the AMP/ATP ratio (11). As well, Ruderman et al. demonstrated that AMPK activity was increased in adipose tissue 30
min following treadmill running exercise in normal rats (30),
suggesting that AMPK is an exercise-inducible factor in adipocytes.
To explore putative factors induced by exercise training to
activate lipolysis, we also examined the effects of compounds
(such as H2O2, sodium-lactate, caffeine, AICAR, and the NO
donor SNAP) that are known to reproduce some of the exercise
A
1.5
Glycerol release
**
SNAP
AICAR
1
**
Caffeine
**
Lactate
0.5
H2O2 short
0
CON
H2O2 long
AEX
Fig. 3. p-NOS/eNOS ratio, p-AMPK/total AMPK ratio, and p-CaMKII/total
CaMKII ratio were examined in adipose tissue of the sedentary control group
(CON, n ⫽ 6) and acute exercise group (AEX, n ⫽ 6) in response to a single
bout of exercise. p-NOS/eNOS and p-AMPK/total AMPK ratios in the AEX
group were significantly higher than those of the CON group. Representative
immunoblots of p-NOS, eNOS, p-AMPK, total AMPK (t-AMPK), p-CaMKII,
and total CaMKII (t-CaMKII) are shown. *P ⬍ 0.05 vs. CON group.
Control
0
200
400
600
800
1000 1200 1400
nmol/mg protein/hr
B
FA release
SNAP
**
AICAR
lating catecholamine (36). These results can be interpreted to
mean that exercise training could protect against mitochondrial
maladaptations observed in adipose tissue of insulin resistance
and T2DM (4, 40). A limitation of the study is that we did not
examine the assumption that augmented mitochondrial oxidative activity in white adipocytes may be required to provide
energy and cyclic AMP for PKA-mediated lipolysis. However,
it is suggested that mitochondrial ATP synthesis is essential for
major metabolic pathways including lipolysis in mature adipocytes [see review in (8)]. In line with this, the higher activity
of the TAG/FA cycle (i.e., lipolysis/re-esterification) in human
visceral white adipose tissue corresponds with relatively high
mitochondrial content and oxidative activity in this fat depot
compared with subcutaneous white adipose tissue [see review
*
Caffeine
Lactate
*
H2O2 short
H2O2 long
Control
0
300
600
900
1200
1500
1800
nmol/mg protein/hr
Fig. 4. Release of (A) glycerol and (B) fatty acids (FA) into the media upon
lipolytic stimulation were measured in differentiated 3T3-L1 cells (n ⫽ 6).
Treatments with sodium-lactate, caffeine or SNAP significantly increased
glycerol release compared with that of the control group. Treatments with 1
mM H2O2, caffeine, or AICAR significantly increased FA release compared
with the control group. *P ⬍ 0.05, **P ⬍ 0.01 vs. control group.
Protein content
(Arbitrary Units)
p-AMPK/
total AMPK
Protein content
(Arbitrary Units)
AEX
•
**
Fig. 5. Expression of mitochondria-related
proteins such as PGC-1␣ and COx were examined in differentiated 3T3-L1 cells that
were pretreated with 10 mM sodium-lactate, 5
mM caffeine, 2 mM AICAR, 100 ␮M SNAP
for 6 h, or 1 mM H2O2 for 15 min (n ⫽ 6).
Treatments with sodium-lactate or SNAP significantly increased expression of PGC-1␣
compared with that of the control group. Treatment with H2O2 significantly increased expression of COx compared with that of the control
group. *P ⬍ 0.05, **P ⬍ 0.01 vs. control
group.
4
*
2
1
0
Protein content
(Arbitrary Units)
Protein content
(Arbitrary Units)
3
*
3
2
1
0
HSL
visceral fat of rats (42). Gaidhu et al. demonstrated that
activating AMPK by AICAR treatment increased the expression
of ATGL, whereas it decreased HSL activity (9). Ogasawara et al.
suggested that the exercise-induced increase in ATGL expression
is regulated through transcriptional activation of the peroxisome
proliferator-activated receptor-␥2 (PPARg2) (27). The present
study provides new insights into mechanisms of the regulation of
LD-associated proteins.
Lactate increased the expression of PGC-1␣ and LD-associated proteins such as HSL, ATGL, perilipin, and CGI-58, all
of which might be potential mechanisms for the increased
lipolysis induced by lactate treatment in this study. Also, a
previous report indicated that lactate activates GPR81, an
orphan G-protein-coupled receptor highly expressed in fat, and
suppresses lipolysis in adipocytes (22). In the present study,
however, cells were incubated with sodium-lactate for 6 h and
3
*
*
*
*
2
1
0
Perilipin
4
3
2
1
0
6
5
4
*
*
**
*
3
2
1
0
CGI-58
*
**
Protein content
(Arbitrary Units)
4
Protein content
(Arbitrary Units)
ATGL
4
3
2
*
**
*
**
Fig. 6. Expression of the LD-associated proteins
such as perilipin, CGI-58, HSL, and ATGL were
examined in differentiated 3T3-L1 cells that
were pretreated with 10 mM sodium-lactate, 5
mM caffeine, 2 mM AICAR, 100 ␮M SNAP for
6 h, or 1 mM H2O2 for 15 min (n ⫽ 6).
Treatments with H2O2, sodium-lactate, caffeine,
or SNAP significantly increased expression of
HSL compared with that of the control group.
Treatments with H2O2, sodium-lactate, caffeine,
or AICAR significantly increased expression of
ATGL compared with that of the control group.
Treatments with sodium-lactate or SNAP significantly increased expression of perilipin compared
with that of the control group. Treatments with
H2O2, sodium-lactate, caffeine, or SNAP significantly increased expression of CGI-58 compared
with that of the control group. *P ⬍ 0.05, **P ⬍
0.01 vs. control group.
1
0
effects in skeletal muscle on levels of LD-associated proteins
and mitochondrial biogenic signaling. Among them, lactate,
ROS, and NO increased levels of mitochondrial biogenic
signaling. Nevertheless, all treatments examined in the present
study increased lipolytic activity assessed by glycerol release
and/or FA release into media in response to lipolytic stimulation. Interestingly, these treatments increased the expression of
LD-associated proteins such as perilipin, CGI-58, HSL, and
ATGL, which regulate lipolysis in a coordinated manner. So
far, few studies have investigated the physiological factors that
increase the expression of LD-associated proteins in adipocytes. For instance, Wohlers and Spangenburg demonstrated
that ovariectomy elevated the interaction of CGI-58 and
ATGL, and decreased perilipin protein content, whereas supplementation with 17␤-estradiol prevented these protein-protein interaction and the reduction of protein content in the
Protein content
(Arbitrary Units)
265
Hashimoto T et al.
COx
PGC1-
Protein content
(Arbitrary Units)
•
266
ACKNOWLEDGMENTS
The authors are grateful to G.C. Henderson for critically reading the
manuscript.
GRANTS
This work was supported in part by a Grant-in-Aid for Young Scientists (B) to
T.H. and K.S., and by a Grant-in-Aid for Young Scientists (A) to M.I. from the
Hashimoto T et al.
Japan Society for Promotion of Science. Partial support was provided by the
Yamaha Motor Foundation for Sports to T.H., the Nakatomi Foundation to T.H.,
and the Suzuken Memorial Foundation to T.H.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: T.H. conception and design of research; T.H. and
K.S. performed experiments; T.H. analyzed data; T.H. interpreted results of
experiments; T.H. prepared figures; T.H. drafted manuscript; T.H. edited and
revised manuscript; T.H., K.S., and M.I. approved final version of manuscript.
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Thus these exercise-inducible factors are attractive therapeutic
effectors against LD-associated metabolic diseases through
activation of lipolysis in adipocytes and of FA oxidation in
muscle.
•
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adipocytes Exercise-inducible factors to activate lipolysis in

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