J Appl Physiol 111: 125–134, 2011.
First published May 5, 2011; doi:10.1152/japplphysiol.00807.2010.
AMPK␣2 deficiency uncovers time dependency in the regulation of
contraction-induced palmitate and glucose uptake in mouse muscle
Marcia J. Abbott,1 Lindsey D. Bogachus,1 and Lorraine P. Turcotte1,2
Departments of 1Biological Sciences and 2Kinesiology, University of Southern California, Los Angeles, California
Submitted 19 July 2010; accepted in final form 27 April 2011
ERK1/2; muscle contraction; caffeine; calcium/calmodulin-dependent
protein kinase kinase; acetyl-CoA carboxylase
cellular signals regulate the
changes in substrate metabolism with muscle contraction, including the AMP-activated protein kinase (AMPK) signaling
cascade (2, 34, 35, 52). During states of low energy, such as
muscle contraction or exercise, it is widely accepted that liver
kinase B1 (LKB1) acts as an upstream AMPK kinase that
phosphorylates and activates AMPK and initiates a multitude
of signaling events to maintain energy homeostasis (39 – 41).
Although strong evidence indicates that the LKB1-AMPK
cascade is a key signaling pathway that regulates changes in
glucose uptake, fatty acid (FA) uptake, and FA oxidation in
contracting skeletal muscle (16, 25, 54, 56), data also show that
AMPK may not be the sole signal mediating contraction-
EVIDENCE SUGGESTS THAT MULTIPLE
Address for reprint requests and other correspondence: L. P. Turcotte, Dept.
of Kinesiology, Dept. of Biological Sciences, College of Letters, Arts, and
Sciences, Univ. of Southern California, 3560 Watt Way, PED 107, Los
Angeles, CA 90089-0652 (e-mail: [email protected]).
http://www.jap.org
induced changes in glucose uptake, FA uptake, and FA oxidation (35, 36, 56).
Along with AMPK, mounting data suggest that increases in
intracellular Ca2⫹ concentration ([Ca2⫹]) may play a key role
in the regulation of glucose uptake, FA uptake, and FA oxidation in skeletal muscle and that this may occur in part via
AMPK activation (3, 28, 51, 53, 56, 57). There is evidence that
Ca2⫹/calmodulin-dependent protein kinase (CaMK) kinase
(CaMKK) activates AMPK in vitro in cells, whole skeletal
muscle, and yeast (17, 20, 21, 55), and we recently showed that
activation of CaMKK and CaMKII leads to activation of
AMPK in perfused rat hindlimbs (1, 35). Additionally, we
showed that inhibition of CaMKK or CaMKII reduces glucose
uptake, FA uptake, and FA oxidation (1, 35). However, it is
unclear whether Ca2⫹ signaling relies extensively on the activation of AMPK to mediate its metabolic effects or whether
there are Ca2⫹-dependent AMPK-independent regulators of
glucose uptake, FA uptake, and FA oxidation. Furthermore, it
is unclear whether AMPK acts upstream of alternative signaling pathways that include extracellular signal-regulated kinases
1 and 2 (ERK1/2) in the regulation of contraction-induced
substrate metabolism. A recent study found that AMPK does
not lie upstream of ERK1/2 following muscle contraction (11);
however, another study measured an increase in ERK1/2 phosphorylation in AMPK transgenic mouse skeletal muscle following treadmill exercise (26).
To study the role of AMPK␣2 in the regulation of glucose
uptake, FA uptake, and FA oxidation in skeletal muscle during
increased intracellular [Ca2⫹] induced by caffeine treatment
and muscle contraction, we employed a transgenic mouse
model possessing a dominant-negative (DN) ␣2-isoform of
AMPK in skeletal muscle and heart muscle (32). Multiple
studies have sought to determine the importance of AMPK in
the regulation of glucose and FA metabolism. However, studies utilizing transgenic mouse models where AMPK is either
inactive or knocked out reveal conflicting results (24 –26, 30).
Some studies suggest that AMPK is necessary in the regulation
of glucose and FA metabolism (24, 25), whereas other studies
show that AMPK is not necessary to measure increases in
substrate metabolism during exercise or other metabolic challenges (26, 30).
Therefore, the purpose of this study was to determine
1) whether a caffeine-induced increase in intracellular [Ca2⫹]
regulates muscle glucose uptake, FA uptake, and FA oxidation
solely via AMPK␣2 activation and 2) whether the ␣2-subunit
of AMPK is essential to increase glucose uptake, FA uptake,
and FA oxidation in contracting skeletal muscle in a hindlimb
perfusion system. Although the perfusion system is not as
physiologically relevant as tracer studies in live animals, it
allowed us to measure glucose uptake, FA uptake, and FA
oxidation at various time points and provided an in situ model.
8750-7587/11 Copyright © 2011 the American Physiological Society
125
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Abbott MJ, Bogachus LD, Turcotte LP. AMPK␣2 deficiency uncovers
time dependency in the regulation of contraction-induced palmitate and
glucose uptake in mouse muscle. J Appl Physiol 111: 125–134, 2011. First
published May 5, 2011; doi:10.1152/japplphysiol.00807.2010.—AMP-activated protein kinase (AMPK) is a fuel sensor in skeletal muscle with multiple
downstream signaling targets that may be triggered by increases in
intracellular Ca2⫹ concentration ([Ca2⫹]). The purpose of this study
was to determine whether increases in intracellular [Ca2⫹] induced by
caffeine act solely via AMPK␣2 and whether AMPK␣2 is essential to
increase glucose uptake, fatty acid (FA) uptake, and FA oxidation in
contracting skeletal muscle. Hindlimbs from wild-type (WT) or
AMPK␣2 dominant-negative (DN) transgene mice were perfused
during rest (n ⫽ 11), treatment with 3 mM caffeine (n ⫽ 10), or
muscle contraction (n ⫽ 11). Time-dependent effects on glucose and
FA uptake were uncovered throughout the 20-min muscle contraction
perfusion period (P ⬍ 0.05). Glucose uptake rates did not increase in
DN mice during muscle contraction until the last 5 min of the protocol
(P ⬍ 0.05). FA uptake rates were elevated at the onset of muscle
contraction and diminished by the end of the protocol in DN mice
(P ⬍ 0.05). FA oxidation rates were abolished in the DN mice during
muscle contraction (P ⬍ 0.05). The DN transgene had no effect on
caffeine-induced FA uptake and oxidation (P ⬎ 0.05). Glucose uptake
rates were blunted in caffeine-treated DN mice (P ⬍ 0.05). The DN
transgene resulted in a greater use of intramuscular triglycerides as a
fuel source during muscle contraction. The DN transgene did not alter
caffeine- or contraction-mediated changes in the phosphorylation of
Ca2⫹/calmodulin-dependent protein kinase I or ERK1/2 (P ⬎ 0.05).
These data suggest that AMPK␣2 is involved in the regulation of
substrate uptake in a time-dependent manner in contracting muscle
but is not necessary for regulation of FA uptake and oxidation during
caffeine treatment.
126
SUBSTRATE METABOLISM DURING MUSCLE CONTRACTION
We hypothesized that AMPK␣2 is necessary to measure increases in glucose and FA uptake, as well as FA oxidation, in
skeletal muscle during a caffeine-induced increase in intracellular [Ca2⫹]. We further hypothesized that AMPK␣2 plays a
role in fuel use in contracting skeletal muscle but that it is not
the sole signal involved in the aforementioned regulation.
MATERIALS AND METHODS
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Animal preparation. Male C57BL/6 mice (24.3 ⫾ 0.80 g body wt,
2–3 mo old) expressing an AMPK␣2 DN transgene (kindly provided
by M. J. Birnbaum, University of Pennsylvania, Philadelphia, PA) in
cardiac and skeletal muscle (32) and their wild-type (WT) littermates
were kept on a 12:12-h light-dark cycle; standard chow and water
were provided ad libitum. Animals were randomly divided into three
experimental groups: rest (n ⫽ 11), caffeine treatment (n ⫽ 10), and
muscle contraction (n ⫽ 11). All procedures for the present study
were approved by the Institutional Animal Care and Use Committee
at the University of Southern California.
Hindlimb perfusion. On the day of the experiment, mice were
anesthetized by an intraperitoneal injection of ketamine-xylazine
cocktail (20 mg/kg body wt). Surgical preparation for the hindlimb
perfusion was performed as previously described (38, 49, 50). Before
placement of the perfusion catheters, heparin (15 IU) was injected into
the inferior vena cava. The mice were then euthanized with an
intracardial injection of pentobarbital sodium (0.4 mg/g body wt), and
catheters were immediately inserted into the descending aorta and
ascending vena cava. The hindlimbs were then washed extensively
with saline and placed in a perfusion apparatus for the equilibration
(20 min) and experimental perfusion (20 min) periods. The perfusion
system employed in this experiment was a reperfusion (36, 38).
The perfusate consisted of Krebs-Henseleit solution, 5% BSA
(Millipore, Billerica, MA), 550 ␮M albumin-bound palmitate (4:1),
24 ␮Ci of albumin-bound [1-14C]palmitate, 6 mM glucose, and 3 mM
caffeine (Sigma, St. Louis, MO) dissolved in Krebs-Henseleit buffer
in the caffeine-treated groups. The perfusate was kept at 37°C and was
continuously gassed with 95% O2-5% CO2. Perfusion flow rate was
maintained at 1.5 ml/min for all groups (average 0.57 ⫾ 0.02
ml·min⫺1·g perfused muscle⫺1). Arterial pH levels were between 7.20
and 7.65, and arterial PO2 and PCO2 were 200 – 455 and 22– 48 mmHg,
respectively. Perfusion pressures were not affected by any of the
experimental conditions and averaged 66.6 ⫾ 16.1, 42.0 ⫾ 12.1, and
66.2 ⫾ 7.2 mmHg in the resting, caffeine-treated, and muscle contraction groups, respectively (P ⬎ 0.05). After the 20-min equilibration period, arterial and venous samples were taken at 5, 10, 15, and
20 min of the experimental period for further analysis. At the end of
the experimental period (20 min), the gastrocnemius-soleus-plantaris
(GSP) muscle groups of both legs were freeze-clamped in situ with
precooled aluminum clamps, removed, and stored in liquid N2 for
further analysis.
Muscle contraction protocol. Force production measurements were
made in anesthetized WT and DN mice to determine whether
AMPK␣2 deficiency would affect (10) or not affect (29) force production with this protocol (13, 14). The right tibiopatellar ligament
was stabilized for the recording of force production, and a modular
chart recorder (Cole Parmer, Vernon Hills, IL) was used to measure
the tension developed by the GSP muscle group during the 20-min
muscle stimulation protocol. The length of the GSP muscle group was
adjusted at the initiation of electrical stimulation to obtain maximal
active tension, and the GSP muscle group was connected to a Grass
stimulator (model S48, Grass Telefactor, West Warwick, RI) to
induce isometric muscle contractions via delivery of 15-V trains of
100 Hz lasting 50 ms with impulse duration of 1 ms with 30
trains/min. This moderate-intensity protocol has been shown to maximize FA metabolism (34).
Perfusate sample analyses. Perfusate samples collected during the
perfusion were analyzed to determine FA, glucose, and lactate con-
centrations, as well as radioactive [14C]FA and 14CO2 contents. A
nonesterified FA kit (NEFA HC kit, WAKO Chemicals, Richmond,
VA) was used to measure perfusate FA concentrations spectrophotometrically. A YSI-1500 analyzer (Yellow Springs Instruments, Yellow Springs, OH) was used to measure glucose and lactate concentrations in the collected perfusate samples. Glycerol concentration was
measured using a glycerol detection reagent (Sigma). Perfusate
[14C]FA and 14CO2 radioactivities were measured as previously
described (46, 48, 49). An ABL-5 analyzer (Radiometer America,
Westlake, OH) was used to determine PCO2, PO2, and pH.
Muscle sample preparation. For Western blot analysis, frozen GSP
muscle samples (40 mg) were powdered under liquid N2 and homogenized in 500 ␮l of ice-cold RIPA buffer, as previously described (34,
36). The total cell homogenate was then transferred to a microcentrifuge tube and vortexed frequently for 1 h, whereupon the samples
were centrifuged at 4,500 g at 4°C for 1 h. For immunoprecipitation
procedures, ⬃90 mg of powdered muscle samples were homogenized
in HEPES buffer and centrifuged at 15,000 g for 5 min. Supernatants
(200 ␮g) were incubated with antibodies for AMPK␣1 or AMPK␣2
(Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h at 4°C with
gentle agitation (35). After the incubation, protein A/G-agarose (Santa
Cruz Biotechnology) was added to the tubes, which were gently
agitated overnight (4°C). The immunoprecipitates were collected by
centrifugation. Pellets were washed with PBS, and the final pellets
were resuspended in sucrose homogenizing buffer and stored at
⫺80°C until analysis. Protein concentrations were determined with
the Bradford protein assay (Bio-Rad, Hercules, CA). Muscle glycogen
content was determined as glucose residues after hydrolysis of the
muscle samples in 1 M HCl (100°C, 2 h) and corrected for free
glucose in the sample (44). For ADP measurements, frozen GSP
muscle samples (10 mg) were powdered under liquid N2 and homogenized as described above. The total cell homogenates were used to
measure ADP levels using a colorimetric assay kit and the manufacturer’s instructions (Abcam, Cambridge, MA). For intramuscular
triglyceride (IMTG) extraction procedures, the Folch method was
utilized as previously described (7, 42). Briefly, ⬃100 mg of muscle
sample were homogenized in 2:1 chloroform-methanol and incubated overnight (4°C) with gentle rotation. NaCl (0.9%) was then
added, and samples were spun at 2,000 g (10 min). The bottom
layer was aspirated, evaporated under N2 gas, and resuspended in
a 1% Triton X-100-PBS solution. The samples were read using a
colorimetric triglyceride (TG) detection reagent (Thermo Scientific, Fremont, CA).
Western blot analysis. Approximately 20 ␮g of protein from the
total cell homogenate preparations were separated on a 10% gel via
SDS-PAGE. Proteins were transferred onto Immobilon-P polyvinylidene difluoride membranes and blocked with 5% BSA in TweenTris-buffered saline for 1 h. The membrane was incubated (4°C) in
5% BSA in Tween-Tris-buffered saline with antibodies (1:1,000)
against phosphorylated (Ser79) acetyl-CoA carboxylase (ACC), total
ACC (Cell Signaling, Danvers, MA), phosphorylated (Thr177)
CaMKI, or total CaMKI (Santa Cruz Biotechnology); phosporylated
ERK1/2, total ERK1/2, or total AS160 (Cell Signaling, Danvers,
MA); or carnitine palmitoyltransferase 1 (CPT1), CD36, peroxisome
proliferator-activated receptor-␥ coactivator 1␣ (PGC1␣), or silent
mating-type information 2 homolog 1 (SIRT1; Santa Cruz Biotechnology). After overnight incubation, the membranes were probed with
a secondary antibody (anti-rabbit IgG; 1:25,000 dilution) raised in
goats (Pierce, Rockford, IL). Blots were then washed and subjected to
enhanced chemiluminescence (Pierce). Band density was quantified
using Scion Image (National Institutes of Health, Bethesda, MD). All
bands analyzed from the perfusion studies were compared with the
band obtained from the resting WT mice. The bands analyzed for
basal protein parameters were compared with a single hindlimb
muscle from an age-matched WT mouse. A Ponceau S total protein
stain (Sigma) was used on the membranes as a loading control.
SUBSTRATE METABOLISM DURING MUSCLE CONTRACTION
RESULTS
Effects of DN transgene on basal protein expression and
force production. In the resting condition, the GSP muscle
group was used to determine whether expression of the DN
transgene led to differences in expression of key proteins
implicated in the regulation of substrate use in skeletal muscle.
There were no alterations in protein content of SIRT1, PGC1␣,
ACC, CPT1, CD36, ERK1/2, CaMKI, or AS160 between WT
and DN mice (Table 1, Fig. 1). Force production by the GSP
Fig. 1. Effect of AMP-activated protein kinase-␣2 (AMPK␣2) dominantnegative (DN) transgene on skeletal muscle protein expression of multiple
signaling intermediates in rest groups from a mixed gastrocnemius-soleusplantaris muscle preparation. Western blots represent total protein content
of silent mating-type information 2 homolog 1 (SIRT1), peroxisome
proliferator-activated receptor-␥ coactivator 1␣ (PCG1␣), acetyl-CoA carboxylase (ACC), carnitine palmitoyltransferase 1 (CPT1), CD36, ERK1/2,
Ca2⫹/calmodulin-dependent protein kinase I (CaMKI), and Akt substrate of
160 kDa (AS160). Bands analyzed for basal protein parameters were
compared with a single hindlimb muscle from an age-matched wild-type
(WT) mouse.
muscle group was not different (P ⬎ 0.05) between the WT
and DN mice (Fig. 2). Force was not produced or recorded in
the resting or caffeine-treated group.
Perfusion characteristics. To verify physiological perfusion
conditions, O2 uptake was measured during the experimental
perfusion period. There were no differences in O2 uptake over
time in any of the experimental groups (P ⬎ 0.05). Caffeine
had no effect on O2 uptake compared with rates measured in
the resting group (P ⬎ 0.05; Table 2). Muscle contraction
resulted in an increase in O2 uptake compared with rest and
Table 1. Effect of AMPK␣2 DN transgene on muscle protein
expression
SIRT1
PGC1␣
ACC
CPT1
CD36
ERK1/2
CaMKI
AS160
WT
DN
106.0 ⫾ 3.0
103.8 ⫾ 3.6
110.1 ⫾ 10.1
101.3 ⫾ 4.6
102.3 ⫾ 0.2
106.3 ⫾ 5.6
123.4 ⫾ 18.2
94.2 ⫾ 5.6
109.5 ⫾ 4.6
114.4 ⫾ 14.7
101.5 ⫾ 7.7
100.4 ⫾ 1.5
107.6 ⫾ 5.0
94.0 ⫾ 3.1
136.4 ⫾ 11.8
108.6 ⫾ 5.5
Values (means ⫾ SE) are expressed as percentage of control of a single
wild-type (WT) hindlimb muscle. Western blotting was performed using
homogenate samples prepared from hindlimbs of WT (n ⫽ 5) or AMPactivated protein kinase (AMPK)-␣2 dominant-negative (DN; n ⫽ 5) mice.
SIRT1, silent mating-type information 2 homolog 1; PGC1␣, peroxisome
proliferator-activated receptor-␥ coactivator 1␣; ACC, acetyl-CoA carboxylase; CPT, carnitine palmitoyltransferase; CAMKI, Ca2⫹/calmodulin-dependent protein kinase I; AS160, Akt substrate of 160-kDa.
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Fig. 2. Effect of AMPK␣2 DN transgene on force production over the 20-min
contraction period. Values are means ⫾ SE (n ⫽ 3 in each group). MC, muscle
contraction.
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Activity assays. AMPK␣1 and AMPK␣2 activities were measured
using [32P]ATP incorporation into SAMS peptide (Upstate Signaling,
Lake Placid, NY), as described elsewhere (36). Briefly, immunoprecipitates were added to an assay cocktail containing [32P]ATP and
SAMS peptide. After incubation, an aliquot was spotted onto a piece
of Whatman filter paper, and all paper samples were washed with
phosphoric acid and then with acetone. Sample papers were analyzed
for radioactivity in a Packard scintillation counter, and counts were
used to calculate phosphotransferase activity.
Calculations and statistics. Palmitate delivery, fractional and total
palmitate uptake, and percent and total palmitate oxidation were
calculated as described previously in detail (46, 49). Acetate correction factors (49) were used to correct percent and total FA oxidation
for label fixation. The specific activity for palmitate in the arterial
samples was not different between groups and did not vary over time,
averaging 153.7 ⫾ 9.8, 160.2 ⫾ 12.6, and 141.9 ⫾ 16.8 ␮Ci/mmol for
the resting, caffeine-treated, and muscle contraction groups, respectively (P ⬎ 0.05). O2 uptake, glucose uptake, and lactate release were
calculated as described elsewhere (49). All uptake and release rates
are expressed per gram of perfused hindlimb muscles of both legs,
which we determined to be ⬃7% of total body weight for WT and DN
mice. The contribution of muscle TG to oxidative metabolism was
calculated on the basis of mean O2 consumption of 2.03 l O2/g fat and
0.746 l O2/g carbohydrate (8, 45). Time effects for glucose, lactate,
glycerol, and FA concentrations and FA kinetic data were analyzed
using a two-way ANOVA with repeated measures for each experimental condition (resting, caffeine-treated, and muscle contraction). If
there were no significant differences in the values measured after 5,
10, 15, or 20 min of perfusion, mean values were used for subsequent
analysis. The effects of experimental condition (resting, caffeine-treated,
and muscle contraction) and genotype (WT vs. DN) were the two factors
analyzed using a two-way ANOVA (Statview 5.0). The Tukey-Kramer
test for post hoc multiple comparisons was performed when appropriate.
A significance level of 0.05 was chosen for all statistical methods.
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SUBSTRATE METABOLISM DURING MUSCLE CONTRACTION
Table 2. Effect of AMPK␣2 DN transgene on hindlimb perfusion characteristics during rest, caffeine treatment, or muscle
contraction
Rest
O2 uptake, ␮mol 䡠 g⫺1 䡠 h⫺1
FA concentration, ␮mol/l
FA delivery, nmol 䡠 min⫺1 䡠 g⫺1
Glucose concentration, mmol/l
Lactate release, ␮mol 䡠 g⫺1 䡠 h⫺1
ADP, ␮mol/g
Caffeine
MC
WT (n ⫽ 5)
DN (n ⫽ 6)
WT (n ⫽ 5)
DN (n ⫽ 5)
WT (n ⫽ 6)
DN (n ⫽ 5)
5.8 ⫾ 1.5
543.4 ⫾ 37.0
337.0 ⫾ 52.2
7.0 ⫾ 0.4
13.2 ⫾ 1.8
0.75 ⫾ 0.04
6.6 ⫾ 1.3
512.7 ⫾ 31.1
301.6 ⫾ 93.9
6.9 ⫾ 0.2
14.3 ⫾ 1.9
0.77 ⫾ 0.10
5.5 ⫾ 1.1
597.5 ⫾ 20.1
375.3 ⫾ 32.3
7.1 ⫾ 0.2
10.3 ⫾ 1.2
0.82 ⫾ 0.05
6.0 ⫾ 0.7
552.2 ⫾ 24.1
391.1 ⫾ 60.3
7.4 ⫾ 0.2
14.3 ⫾ 1.3
0.81 ⫾ 0.10
11.4 ⫾ 0.8*†
604.3 ⫾ 47.5
413.7 ⫾ 66.0
7.2 ⫾ 0.1
36.7 ⫾ 4.9*†
0.88 ⫾ 0.10
10.3 ⫾ 1.6*†
541.8 ⫾ 34.8
337.8 ⫾ 28.7
6.7 ⫾ 0.3
30.3 ⫾ 5.2*†
0.75 ⫾ 0.03
Values are means ⫾ SE; n, number of mice. WT or AMPK␣2 DN mice were perfused during rest, caffeine treatment, or moderate-intensity muscle contraction
(MC). *P ⬍ 0.05 vs. respective rest group. †P ⬍ 0.05 vs. caffeine group.
3C). During muscle contraction, time-dependent differences
were uncovered between the WT and DN mice. Within 5 min
of initiation of the muscle contraction protocol, glucose uptake
had increased by 156% in the WT mice, and this elevated
glucose uptake rate was maintained throughout the rest of the
contraction period (P ⬎ 0.05; Fig. 3A). In contrast, glucose
uptake rates were not elevated in the DN mice until the last 5
min (200% increase vs. resting DN mice at the same time
point) of muscle contraction (P ⬍ 0.05; Fig. 3A). At rest and
during caffeine treatment, glucose uptake did not vary over
time (Fig. 3B). At rest, muscle glycogen content was 29%
lower in the DN than WT group (P ⬍ 0.05; Fig. 3D). In the
WT mice, caffeine treatment and muscle contraction resulted
in a 33–38% reduction in muscle glycogen content (P ⬍ 0.05;
Fig. 3D). In the DN mice, muscle glycogen content was not
affected by caffeine treatment or muscle contraction (P ⬎
0.05). During muscle contraction, glucose uptake, expressed as
percentage of its respective rest group, was lower in the DN
than WT mice at 5, 10, and 15, but not 20, min (P ⬍ 0.05) and
Fig. 3. Effect of AMPK␣2 DN transgene on
time effects for glucose uptake in muscle
contraction (A) and caffeine treatment (B)
groups and mean glucose uptake (C) and
glycogen content (D) in perfused mouse
hindlimbs during caffeine (Caff) treatment or
moderate-intensity muscle contraction. R,
rest. Values are means ⫾ SE (n ⫽ 5 for R ⫹
WT, Caff ⫹ WT, Caff ⫹ DN, and MC ⫹
DN; n ⫽ 6 for R ⫹ DN and MC ⫹ WT).
*P ⬍ 0.05 vs. respective rest group; #P ⬍
0.05 vs. respective WT group; &P ⬍ 0.05 vs.
rest ⫹ WT; ##P ⬍ 0.05 vs. caffeine groups;
**P ⬍ 0.05 vs. MC ⫹ DN group; †P ⬍ 0.05
vs. 5 min; ***P ⬍0.05 vs. 10 min.
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caffeine treatment (P ⬍ 0.05). For all experimental conditions,
O2 uptake was not significantly different between WT and DN
mice (P ⬎ 0.05). Palmitate concentration and delivery were not
significantly different at any time point in any of the groups, as
dictated by the protocol (P ⬎ 0.05; Table 2). In WT and DN
mice, muscle ADP levels were not affected (P ⬎ 0.05) by
caffeine treatment or muscle contraction. Similarly, the DN
transgene had no effect on muscle ADP levels (P ⬎ 0.05;
Table 2).
Substrate exchange across the hindlimb. Lactate release was
increased (178%) during muscle contraction (P ⬍ 0.05; Table
2). The DN transgene did not affect lactate release in any of the
groups (P ⬎ 0.05). Arterial perfusate glucose concentration did
not vary over time in any of the groups, and mean glucose
concentration was not different between any of the groups
(P ⬎ 0.05; Table 2). Mean glucose uptake (␮mol·h⫺1·g⫺1) was
significantly increased (P ⬍ 0.05; Fig. 3C) by caffeine treatment (168%), and the DN transgene blunted the caffeineinduced increase in glucose uptake by 34% (P ⬍ 0.05; Fig.
SUBSTRATE METABOLISM DURING MUSCLE CONTRACTION
tended to increase (P ⫽ 0.089) over time in the DN mice
(Fig. 5A).
Palmitate metabolism. At rest and during caffeine treatment,
palmitate uptake (nmol·min⫺1·g⫺1) did not vary over time
(P ⬎ 0.05; Fig. 4, A and B). Mean palmitate uptake was
significantly increased (P ⬍ 0.05; Fig. 4E) by caffeine treatment (54%), but there were no DN effects. As shown for
glucose uptake, time-dependent differences were found between the WT and DN mice during muscle contraction. Within
5 min of initiation of the muscle contraction protocol, palmitate
uptake had increased 76% and 54% in the WT and DN mice,
respectively, over their resting counterparts, and palmitate
uptake remained elevated in the WT mice (P ⬍ 0.05; Fig. 4A).
However, the contraction-induced elevation in palmitate up-
129
take had dropped off after 10 min in the DN mice, and
palmitate uptake was not different from the resting condition
for the remaining 10 min of the muscle contraction protocol
(P ⬎ 0.05; Fig. 4A). During muscle contraction, palmitate
uptake, expressed as percentage of its respective rest group,
was lower in the DN than WT mice at 20 min (P ⬍ 0.05; Fig. 5B).
In WT mice, mean palmitate oxidation (nmol·min⫺1·g⫺1; Fig.
4F) increased by 180% with caffeine treatment and 260% with
muscle contraction. In the resting condition, palmitate oxidation was higher (120%) in the DN than WT group (P ⬍ 0.05;
Fig. 4F). Unlike glucose uptake and palmitate uptake, no
time-dependent effects were found in the caffeine or muscle
contraction condition (P ⬎ 0.05; Fig. 4, C and D). Mean
contraction-mediated palmitate oxidation was blunted in the
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Fig. 4. Effect of AMPK␣2 DN transgene on
time effects for palmitate uptake in muscle
contraction (A) and caffeine treatment (B)
groups, time effects for palmitate oxidation
in muscle contraction (C) and caffeine treatment (D) groups, and mean palmitate uptake
(E), mean palmitate oxidation (F), mean
glycerol release (G), and triglyceride (TG)
content expressed as percent decrease compared with average respective rest group (H)
in perfused mouse hindlimbs during caffeine
treatment or moderate-intensity muscle contraction. Values are means ⫾ SE (n ⫽ 5 for
rest ⫹ WT, caffeine ⫹ WT, caffeine ⫹ DN,
and MC ⫹ DN; n ⫽ 6 for rest ⫹ DN and
MC ⫹ WT). *P ⬍ 0.05 vs. respective rest
group; #P ⬍ 0.05 vs. respective WT group;
&P ⬍ 0.05 caffeine effect; **P ⬍ 0.05 vs.
MC ⫹ DN group; †P ⬍ 0.05 vs. 5 min.
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Fig. 5. Effect of AMPK␣2 DN transgene on glucose uptake (A) and palmitate
uptake (B). Values are means ⫾ SE (n ⫽ 6 for MC ⫹ WT; n ⫽ 5 for MC ⫹
DN). #P ⬍ 0.05 vs. WT group.
DN mice and not significantly different from the resting condition (P ⬎ 0.05; Fig. 4F). Mean glycerol release throughout
the perfusion period was not significantly different in any of
the perfusion conditions; however, during rest and muscle
contraction, glycerol release was higher in the DN groups than
their respective WT groups (P ⬍ 0.05; Fig. 4G). Mean muscle
TG content was not different between the WT and DN groups
during the resting condition (P ⬎ 0.05, 19.9 ⫾ 1.9 ␮mol/g).
After muscle contraction, TG content was lower in WT and
DN mice. However, the DN group had 39% less IMTG, while
the WT group had 20% less IMTG, during muscle contraction
(P ⬍ 0.05; Fig. 4H). Neither caffeine nor genotype had an
effect on muscle TG content.
Enzyme activities. As expected, AMPK␣2 activity (pmol·
min⫺1·g⫺1) was decreased by the DN transgene (P ⬍ 0.05;
Fig. 6A). Caffeine treatment and muscle contraction increased
(P ⬍ 0.05; Fig. 6A) AMPK␣2 activity by 81% and 161%,
respectively, but did not affect (P ⬎ 0.05) AMPK␣1 activity (Fig.
6B). The DN transgene completely prevented the increase in
AMPK␣2 activity with muscle contraction and caffeine treatment
(Fig. 6A) but did not affect (P ⬎ 0.05) AMPK␣1 activity (Fig.
6B). Total protein expression of ACC was not affected by caffeine
treatment or muscle contraction, and there was no effect of the DN
transgene (P ⬎ 0.05; Fig. 6C). In the WT mice, caffeine treatment
J Appl Physiol • VOL
Fig. 6. Effect of AMPK␣2 DN transgene on AMPK␣2 activity (A), AMPK␣1
activity (B), and ACC phosphorylation (C) in perfused mouse hindlimbs during
caffeine treatment or moderate-intensity muscle contraction. Activity values
were calculated per gram of muscle present in assay preparation (AMPK) or
per microgram of protein present in assay preparation. Preparations are from
the mixed gastrocnemius-soleus-plantaris muscle group. p-ACC (Ser79), phosphorylated (Ser79) ACC. Values are means ⫾ SE (n ⫽ 5 in each group). *P ⬍
0.05 vs. rest ⫹ WT group; #P ⬍ 0.05 vs. respective WT group.
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SUBSTRATE METABOLISM DURING MUSCLE CONTRACTION
and muscle contraction increased (P ⬍ 0.05) phosphorylation of
ACC by 32% and 64%, respectively (Fig. 6C). Consistent with the
results obtained for AMPK␣2 activity, the DN transgene prevented the increase in phosphorylated ACC with caffeine treatment and muscle contraction. In the rest condition, phosphorylated ACC was 11% lower in the DN than WT mice.
Activation of signaling intermediates. Total protein expression of CaMKI and ERK1/2 was not different between any of
the groups (P ⬎ 0.05), and there was no effect of the DN
transgene for any of the proteins (Fig. 7). Phosphorylation
(Thr177) of CaMKI was increased by 150% and 104% with
caffeine treatment and muscle contraction, respectively (P ⬍
0.05; Fig. 7A). ERK1/2 phosphorylation was not affected by
caffeine treatment (P ⬎ 0.05; Fig. 7B) but was increased 38%
by muscle contraction (P ⬍ 0.05; Fig. 7B).
J Appl Physiol • VOL
DISCUSSION
Our data provide further evidence for the involvement of
AMPK␣2 in the regulation of substrate use during muscle
contraction. Additionally, the results of this study suggest that
while AMPK␣2 is activated by caffeine-induced increases in
intracellular [Ca2⫹], this activation is not essential to observe
an increase in substrate uptake and FA oxidation during caffeine treatment. Additionally, these data indicate a role for
AMPK-independent Ca2⫹-dependent signaling in the regulation of substrate metabolism in skeletal muscle. However,
AMPK␣2 appears to be necessary to record normal timedependent changes in FA and glucose uptake in contracting
mouse skeletal muscle, and these findings are not in agreement
with previous studies that showed that AMPK activation is not
time-dependent (27, 36). Indeed, one of the more interesting
findings of this study is that AMPK␣2 deficiency led to a
reciprocal switch in FA and glucose uptake during the 20-min
contraction protocol.
Interestingly, during resting perfusion conditions, FA oxidation rates were elevated in mice that possessed the AMPK␣2
DN transgene. These results were surprising to us, since
AMPK␣2 has been repeatedly shown to be an upstream regulator of ACC activation, which in turn has been shown to
reduce malonyl-CoA content, resulting in a rise in FA oxidation rate (37, 52). We hypothesized that low AMPK␣2 activity
measured in the DN mice would result in lower or equivalent
resting FA oxidation rates, as well as lower ACC phosphorylation, than in WT mice. In line with our hypothesis, we
measured a decrease in ACC phosphorylation during the resting condition in the DN mice, which is consistent with previous findings (26), but this decrease in ACC phosphorylation
was not accompanied by a decrease in FA oxidation. This is in
agreement with recent reports demonstrating that ACC may not
be the only regulator of FA oxidation (33). We attempted to
determine if the DN mice had increased expression of proteins
known to be associated with the regulation of FA oxidation in
skeletal muscle, such as CD36, CPT1␤, and PGC1␣, which
may have accounted for the increase in oxidation at rest.
However, no differences in protein content were observed.
Because we could not isolate mitochondrial membrane fractions on top of nuclear fractions from the small amount of
hindlimb muscles that we had, we could not determine whether
CD36 protein content was altered in mitochondrial membranes. Indeed, it has been suggested by some (18, 19), but not
others (23), that mitochondrial membrane CD36 content is an
important factor in the regulation of FA oxidation. Because of
the methodological limitations described above, we cannot
contribute data to this debate. Given that AMPK␣2 is known to
regulate gene expression (15), the deficiency in AMPK␣2
activity associated with our model was likely associated with
alterations in the expression of genes involved in metabolic
regulation in DN mice. This conclusion is consistent with the
possibility that multiple redundant pathways are responsible
for regulating substrate metabolism in skeletal muscle.
It has been shown by us and others that caffeine-induced
increases in Ca2⫹ signaling regulate substrate metabolism,
possibly via AMPK activation (1, 6, 22, 35). Indeed, we have
shown that when CaMKII or CaMKK was inhibited during
caffeine stimulation, there were subsequent decreases in
AMPK␣2 activity along with decreases in the rates of FA
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Fig. 7. Effect of AMPK␣2 DN transgene on CaMKI (A) and ERK1/2 (B)
protein expression and phosphorylation state in perfused mouse hindlimbs
during caffeine treatment or moderate-intensity muscle contraction. Preparations are from the mixed gastrocnemius-soleus-plantaris muscle group.
CaMKI-p (Thr177), phosphorylated (Thr177) CaMKI; ERK1/2-p, phosphorylated ERK1/2. Values are means ⫾ SE (n ⫽ 5 in each group). *P ⬍ 0.05 vs.
respective rest group.
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SUBSTRATE METABOLISM DURING MUSCLE CONTRACTION
J Appl Physiol • VOL
during muscle contraction or treadmill exercise (24, 25). However, our data are not consistent with some additional reports in
which low AMPK␣2 activity did not affect muscle glucose
clearance during treadmill exercise (26), glucose uptake in
contracting extensor digitorum longus muscle, which is a
mixed-fiber type muscle (10), or FA oxidation in isolated
muscle during muscle contraction or following treadmill exercise (4, 10, 25, 29, 30). Possible explanations for these differences include the fact that the exercise mode and the fiber types
were different between studies. In the current study, we utilized
an in situ electrical stimulation protocol and measured substrate uptake and FA oxidation during moderate-intensity muscle contraction in a mixed-muscle preparation. In contrast,
Miura et al. (30) measured FA oxidation in incubated muscle
strips following a low-intensity treadmill exercise protocol. As
such, their data might be more appropriately viewed as postexercise data. Similarly, it is generally recognized that incubated
muscle strips, such as those used by Dzamko et al. (4), lack
proper circulation, and, as such, the response of these preparations is not always similar to that of perfused muscle. On the
other hand, low AMPK␣2 activity did not affect muscle glucose clearance during treadmill exercise in chronically cannulated mice whose muscle maintains functional circulation and
appropriate neural input (26). Most importantly, our data are
different from results reported by others, because the perfused
hindlimb model allowed us to measure substrate uptake and FA
oxidation at different times during the muscle contraction
protocol. Time-dependent analysis revealed that decreased
AMPK␣2 activity impaired the ability of the contracting muscle to increase glucose uptake during the early phase of the
stimulation protocol and to maintain higher rates of FA uptake
throughout the stimulation protocol. However, as shown in
isolated muscle strips (10), our glucose uptake rates were not
affected by low AMPK␣2 activity during the last 5 min of the
stimulation protocol. This comparison demonstrates that the
timing of the measurements is important when studying cellular signaling during muscle contraction. Consistent with previous data in the same mouse model (31, 32), DN mice had
lower resting muscle glycogen content and did not utilize
glycogen to the same extent as the WT mice during muscle
contraction. However, this is not consistent with recent reports
suggesting similar or higher rates of glycogen utilization during treadmill exercise in muscle of mice with low AMPK␣2
activity (24, 26). Given that glycogen use and mean glucose
uptake and FA oxidation were lower in the DN mice during
muscle contraction, alternate fuel sources must have been used
to maintain force production. Consistent with this notion, the
glycerol and TG data show that contracting muscle in DN mice
relied more heavily on intramuscular TG as a fuel source.
Evidence shows that AMPK phosphorylates and inhibits hormone-sensitive lipase at Ser565 in skeletal muscle and that this
is associated with a reduced rate of lipolysis (12). Given the
absence of AMPK␣2 activation in the DN mice, our results
agree with these other data and are consistent with an increase
in the lipolytic rate and a greater reliance on muscle TG as a
fuel. To ascertain that the measured TG breakdown would be
sufficient as a fuel source to supply alternate fuel in the DN
mice, we estimated the contribution of muscle TG to total
oxidative metabolism in DN mice. Given an average O2 uptake
of 10.3 ␮mol·g⫺1·h⫺1 in the DN mice, our calculations estimate that ⬍1 ␮mol of muscle TG would have been needed to
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uptake, glucose uptake, and FA oxidation (1, 35). In line with
these previous results, we measured an increase in AMPK␣2
activity in caffeine-treated WT mice in this study. Because
caffeine-induced AMPK␣2 activity was reduced in the DN
mice, we expected to measure coincident decreases in FA
uptake and oxidation with caffeine treatment. However, there
were no measurable differences between the DN and WT mice
in FA metabolism during caffeine treatment.
In contrast, caffeine-induced glucose uptake was blunted in
the DN mice. Although glucose uptake was lower with caffeine
treatment in the DN mice, there continued to be elevated
glucose uptake rates over resting conditions. This increase in
glucose uptake supports the notion that glucose uptake is
regulated by Ca2⫹ signaling intermediates that are independent
of AMPK activation (22, 53). Our results further suggest that
AMPK␣2 activation may be more critical to the regulation of
glucose metabolism during states of increased intracellular
[Ca2⫹] induced by caffeine than to the regulation of FA
metabolism in skeletal muscle. It has also been postulated by
some that AMPK activation is associated with increased expression of a downstream target of the CaMKK pathway, such
as CaMKIV, during low energy states (58). However, we did
not measure any effect of the DN transgene on the expression
or phosphorylation state of CaMKI, an additional downstream
target of CaMKK (5, 43), in any of the experimental conditions. These data suggest that CaMKK signaling either lies
upstream of AMPK␣2 or is not affected by low AMPK␣2
activity. While no caffeine-induced force production was observed in our study, the increase in Ca2⫹ release from the
sarcoplasmic reticulum induced by caffeine treatment may
have been associated with a small twitch potentiation that was
unrecordable with our system. We consider this possibility to
be unlikely, because caffeine-induced twitch potentiation and
force production have not been measured at concentration
⬍3.5 mM in mixed skeletal muscle (9, 57). It should be noted
that there was variation in the concentration of FA in the
perfusate preparation. Unlike glucose and pyruvate, for example, controlling for albumin-bound FA concentration in perfusate is challenging. This is due mostly to the fact that cold and
radioactive FA (in this study, palmitate) is bound to albumin in
separate batches before they are introduced to the perfusion
medium on the day of the experiment. However, given that FA
availability can affect FA uptake under some, but not all,
physiological conditions (45, 46), we wanted to ensure that the
variability in palmitate concentration was not associated with
our calculated values for palmitate uptake. Correlational analysis of palmitate concentration vs. palmitate uptake resulted in
a low association (R2 ⫽ 0.06, P ⬎ 0.05; data not shown),
suggesting that the variation in the palmitate concentration did
not affect our palmitate uptake measurements. Taken together,
our results suggest that caffeine-induced AMPK␣2 activation is
important in the regulation of glucose uptake but is not required to measure increases in FA uptake and oxidation in
skeletal muscle during caffeine treatment.
Because significant metabolic shifts were observed during
muscle contraction in the DN mice, our data suggest that
appropriate AMPK␣2 activation is necessary to measure a
typical response in glucose uptake, FA uptake, and FA oxidation during muscle contraction. These conclusions are consistent with previous studies in which AMPK␣2 was shown to be
a necessary signal in the regulation of substrate metabolism
SUBSTRATE METABOLISM DURING MUSCLE CONTRACTION
ACKNOWLEDGMENTS
The authors thank Emily Kallen, Matthew Burkhard, and Nadia El-Fakih
for valuable technical expertise.
GRANTS
This study was supported by grants from the University of Southern
California Women in Science and Engineering Program and by fellowships
from the Integrative and Evolutionary Biology Program and the Gold Family
Foundation.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
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