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Enhanced muscle insulin sensitivity after contraction/exercise is mediated by AMPK

Rasmus Kjbsted1,2, Nanna Munk-Hansen1, Jesper B. Birk1, Marc Foretz3,4,5, Benoit

Viollet3,4,5, Marie Bjrnholm6, Juleen R. Zierath2,6, Jonas T. Treebak2, Jrgen F.P.
Wojtaszewski1
1

Section of Molecular Physiology, Department of Nutrition, Exercise and Sports, Faculty of

Science, University of Copenhagen, DK-2100 Copenhagen, Denmark
2

Section of Integrative Physiology, Novo Nordisk Foundation Center for Basic Metabolic

Research, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200

Copenhagen, Denmark
3

INSERM, U1016, Institut Cochin, Paris, France

4

5
6

CNRS, UMR8104, Paris, France

Universit Paris Descartes, Sorbonne Paris Cit, Paris, France

Integrative Physiology, Department of Molecular Medicine and Surgery, Karolinska Institutet,

Stockholm, Sweden
Short running title: AMPK and muscle insulin sensitivity
Key words: exercise, glucose uptake, TBC1D4, AS160, AMP-activated protein kinase
Figures / Tables: 8 / 0
Word count: 5058
References: 56
Corresponding author:
Jrgen F.P. Wojtaszewski, PhD
Section of Molecular Physiology
Department of Nutrition, Exercise and Sports
University of Copenhagen
Universitetsparken 13
DK-2100, Copenhagen, Denmark
Phone: +45 3532 1625
Email: jwojtaszewski@nexs.ku.dk

Diabetes Publish Ahead of Print, published online October 26, 2016

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Abstract
Earlier studies have demonstrated that muscle insulin sensitivity to stimulate glucose uptake is
enhanced several hours after an acute bout of exercise. Using 5-aminoimidazole-4-carboxamideribonucleotide (AICAR), we recently demonstrated that prior activation of AMPK is sufficient to
increase insulin sensitivity in mouse skeletal muscle. Here we aimed to determine whether
activation of AMPK is also a prerequisite for the ability of muscle contraction to increase insulin
sensitivity. We found that prior in situ contraction of m. extensor digitorum longus (EDL) and
treadmill exercise increased muscle and whole body insulin sensitivity in wild type (WT) mice,
respectively. These effects were not found in AMPK12 muscle-specific knockout mice. Prior in
situ contraction did not increase insulin sensitivity in m. soleus from either genotype. Improvement
in muscle insulin sensitivity was not associated with enhanced glycogen synthase activity or
proximal insulin signaling. However, in WT EDL muscle prior in situ contraction enhanced insulinstimulated phosphorylation of TBC1D4 Thr649 and Ser711. Such findings are also evident in prior
exercised and insulin sensitized human skeletal muscle. Collectively, our data suggest that the
AMPK-TBC1D4 signaling axis is likely mediating the improved muscle insulin sensitivity after
contraction/exercise and illuminates an important and physiological relevant role of AMPK in
skeletal muscle.

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Introduction
Skeletal muscle from both human, sheep, dog, and rodents demonstrates increased insulinstimulated glucose uptake in the period after a single bout of exercise (110). This phenomenon is
observed in both normal and insulin-resistant muscle (1113) and has been suggested to involve an
increased abundance of GLUT4 at the plasma membrane (14). Moreover, changes in muscle insulin
sensitivity occurs independent of changes in protein synthesis (15), indicating involvement of
posttranslational mechanisms. Interestingly, studies of human and rodent muscle suggest that prior
exercise does not improve the ability of insulin to stimulate components of the proximal insulin
signaling cascade including the insulin receptor, insulin receptor substrate 1, PI3K, and Akt (5,15
18). This supports the notion that improved insulin sensitivity after prior exercise is not caused by
enhanced delivery of insulin to the muscle and indicates an important role for more distal
intramyocellular signaling events.
AMP-activated protein kinase (AMPK) is a heterotrimeric complex containing
catalytic and regulatory and subunits of which several isoforms exist (1, 2, 1, 2, 1, 2
and 3) (19). In human and mouse skeletal muscle, 3 (221, 223 and 121) and 5 (221,
223, 211, 121 and 111) heterotrimeric combinations have been found, respectively
(20,21). Interestingly, mouse skeletal muscle contains two 1-associated complexes that are not
found in human skeletal muscle. Furthermore, in mouse EDL and human vastus lateralis muscle,
the 223 complex represents ~20% of all AMPK heterotrimer complexes, whereas in mouse SOL
muscle it comprises less than 2% (20,21). AMPK is considered an important sensor of cellular
energy balance, and in skeletal muscle AMPK is activated during conditions of cellular stress such
as muscle contraction and hypoxia (22). When activated, AMPK stimulates ATP generating
processes (e.g., glucose uptake and lipid oxidation) while inhibiting ATP consuming processes
(e.g., protein and lipid synthesis) in an attempt to restore cellular energy homeostasis (22,23).

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TBC1D4 is phosphorylated by multiple kinases (including Akt) during insulin

stimulation (24,25). This modification has been suggested to be important for insulin-stimulated
glucose uptake (26). AMPK is also upstream of TBC1D4, and both contraction- and AICARinduced AMPK activation increase phosphorylation of TBC1D4 (25). Within recent years,
TBC1D4 has emerged as a likely candidate for mediating the insulin-sensitizing effect of prior
exercise on skeletal muscle glucose uptake. In support of this, phosphorylation of TBC1D4 is
elevated in prior exercised human and rat muscle, concomitant with enhanced insulin sensitivity
(11,17,18,2729).
Prior AICAR stimulation increases insulin sensitivity to stimulate glucose transport in
rat muscle (15), and we have recently provided evidence that this is mediated by AMPK in muscle
of mice (30). We also reported a positive association between insulin-stimulated glucose uptake and
phosphorylation of regulatory sites on TBC1D4 (30). This suggests a mechanism by which AICAR,
through AMPK, potentiates a subsequent effect of a submaximal concentration of insulin on
TBC1D4 leading to improved insulin-stimulated glucose uptake.
During AICAR stimulation, cells maintain energy and fuel homeostasis. In contrast,
the myocyte is subjected to energy and fuel disturbances during exercise/contraction which likely
contributes to AMPK activation. Furthermore, while AMPK regulates muscle glucose uptake, fatty
acid uptake, gene activation, and mitochondrial protein content in response to AICAR treatment
(31-34), activation of AMPK is not necessary for inducing such effects in response to
exercise/contraction (31-36). Hence, little evidence exits to support the assumption that AICARand exercise/contraction-induced biological responses are equally dependent on AMPK activation.
Since the first proposal of an insulin-sensitizing effect of prior exercise by Bergstrm
and Hultman (37) and the subsequent proof of this in rat and human skeletal muscle (1,2), an
ongoing search for molecular interactions between exercise and insulin signaling has occurred. To

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further study this, we established an experimental protocol in which mouse muscle displays
enhanced insulin sensitivity to stimulate glucose uptake after in situ contraction. We used this
model to provide genetic evidence for the hypothesis that AMPK acts as a molecular transducer
between exercise and insulin signaling and thus, is necessary for the ability of prior
contraction/exercise to increase muscle insulin sensitivity.

Research Design and Methods

Animals
All experiments were approved by the Danish Animal Experiments Inspectorate (#2014-15-293401037; #2013-15-2934-00911), as well as the regional ethics committee of Northern Stockholm and
complied with the EU convention for protection of vertebra animals used for scientific purposes
(Council of Europe, Treaty 123/170, Strasbourg, France, 1985/1998). Animals used in this study
were AMPK12 muscle-specific double KO and whole body AMPK3 KO female mice with
corresponding WT littermates as controls (35,36,38). Animals (16 wks. 5 SD) were maintained on
a 12:12 light-dark cycle (6:00AM-6:00PM) with unlimited access to standard rodent chow and
water.

Glucose uptake during in situ contraction of EDL and SOL muscle

For all experiments, fed mice were anesthetized by an intraperitoneal injection of pentobarbital (10
mg/100 g body weight) before both common peroneal and tibial nerves were exposed.
Subsequently, an electrode was placed on a single common peroneal or tibial nerve followed by in
situ contraction of extensor digitorum longus (EDL) or soleus (SOL) muscle, respectively. The
contralateral leg served as a rested control. The contraction protocol consisted of 0.5-sec. trains
(100 Hz, 0.1 ms, 2-5V) repeated every 1.5 sec. for 10 min. To determine glucose uptake during in

Diabetes

situ contraction, tail blood were collected at time points 0, 5, and 10 min. Following the first blood
sample, a bolus of [3H]2-deoxyglucose (12.3 MBq/kg body wt) dissolved in isotonic saline was
injected retroorbitally. After the last blood sample, EDL or SOL muscles were rapidly dissected and
frozen in liquid nitrogen. Uptake of [3H]2-deoxyglucose into muscle was assessed based on
accumulated [3H]2-deoxyglucose-6-phosphate and tracer specific activity in plasma as previously
described (39).

Muscle insulin sensitivity following in situ contraction

For measurements of insulin sensitivity following in situ contraction, electrodes were connected to
either the common peroneal nerve (EDL) or the tibial nerve (SOL) of both legs of the anesthetized
animals. One half of the animals served as sham-operated controls. Immediately after in situ
contraction of EDL or SOL, muscles were dissected and suspended at low tension (~1 mN) in
incubation chambers (Model 610/820M, Danish Myo-Technology, Denmark) containing KrebsRinger-Buffer (KRB) [117 mmol/L NaCl, 4.7 mmol/L KCl, 2.5 mmol/L CaCl2, 1.2 mmol/L
KH2PO4, 1.2 mmol/L MgSO4, 0.5 mmol/L NaHCO3 (pH 7.4)] supplemented with 0.1% BSA, 5
mmol/L mannitol and 5 mmol/L D-glucose. During the entire incubation period, the buffer was
oxygenated with 95% O2 and 5% CO2 and maintained at 30C. SOL and EDL muscles were
allowed to recovery for 2 and 3 hours, respectively. These time points were selected based on
measurements demonstrating reversal of muscle glucose uptake following in situ contraction.
During recovery, the incubation medium was replaced once every 30 min to maintain an adequate
glucose concentration. Subsequently, basal, submaximal (100 U/mL / 694.5 pmol/L) and maximal
(10,000 U/mL / 69450 pmol/L; only EDL) insulin-stimulated 2-deoxyglucose uptake was
measured during the last 10 min of a 30 min stimulation period by adding 1 mmol/L [3H]2deoxyglucose (0.028 MBq/mL), 7 mmol/L [14C]mannitol (0.0083 MBq/mL) and 2 mmol/L

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pyruvate to a glucose-free incubation medium. 2-deoxyglucose uptake was assessed by the

accumulation of [3H]2-deoxyglucose into muscle with the use of [14C]mannitol as an extracellular
marker (30). Radioactivity was measured on 200 L lysate by liquid scintillation counting (Ultima
GoldTM and Tri-Carb 2910 TR, Perkin Elmer) and related to the specific activity of the incubation
media.

Post-exercise insulin tolerance test and in vivo muscle glucose uptake

All mice were acclimatized to treadmill running on 5 consecutive days. The acclimatization
consisted of a 2 min warm up (0-10.2 m/min) followed by 5 minutes of running at 10.2 m/min and
0 incline. 2 days after the acclimatization mice were subjected to a graded maximal running test as
previously described (36). For insulin tolerance tests (ITT), mice were fasted in single cages for 2
hours (~8:00-10:00AM) before performing a single bout of treadmill exercise (30 min, 15 incline
and 55% of maximal running speed). Resting control mice were left in the cage. Following exercise,
mice were returned to their individual cage without access to food for 1 hour after which they were
administered with either 0.3U (2.09 nmol) or 0.4U (2.78 nmol) of insulin per kg body weight
intraperitoneally, respectively. Throughout the ITT, blood was collected from the tail vein at 0, 20,
40, 60, 90, and 120 min and blood glucose concentration was determined using a glucometer
(Contour XT, Bayer, Leverkusen, Germany). Area over the curve values were calculated from time
points 0 to 40 min, since changes in blood glucose concentrations at later time points may largely
reflect the ability to counteract hypoglycaemia rather than peripheral glucose disposal. 3-4 weeks
after the last ITT, in vivo muscle glucose uptake during the first 40 min of an ITT (0.3 U/kg insulin)
was measured in the same mice 1 h after treadmill exercise. All mice received an intraperitoneal
injection of insulin dissolved in isotonic saline (10 l / g body weight) containing 0.1 mM [3H]2deoxyglucose (1,78 MBq) 1 h after rest and exercise. Immediately before, as well as 20 and 40 min

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into the ITT blood glucose concentration was measured from the tail vein and blood samples were
obtained for determination of radioactivity. Following the last blood sample, mice were euthanized
by cervical dislocation and tissues were rapidly dissected and frozen in liquid nitrogen. Uptake of
[3H]2-deoxyglucose into muscle was assessed as described above (39).

Ex vivo contraction of incubated skeletal muscle

Whole body AMPK3 KO mice were anesthetized and EDL muscles were isolated and
preincubated in KRB (40 min) before stimulated to contract (10 min) as previously described (40).

Muscle processing
Muscles were homogenized in 400 L ice-cold homogenization buffer (30) and rotated end-overend for 1 h at 4 C. Part of the homogenate was centrifuged at 16,000g for 20 min at 4 C after
which lysate (supernatant) was collected and frozen in liquid nitrogen for later analyses. Total
protein abundance in muscle lysate and homogenate was determined by the bicinchoninic acid
method (ThermoFisher Scientific, Waltham, MA).

Glycogen synthase activity

Muscle glycogen synthase (GS) activity was measured in 75 g muscle homogenate using 96 well
microtitre plates as previously described (11,41). Samples were assayed in triplicate in the presence
of 0.02 and 8.0 mmol/L glucose-6-phosphate and presented as per cent glucose-6-phosphate
independent activity (GS0.02*100/GS8.0; %I-form) and total glycogen synthase activity (GS8.0; total),
respectively.

AMPK activity

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Heterotrimer-specific AMPK activity in mouse skeletal muscle was determined as previously

described (30). AMPK activity was measured on 300 g of muscle lysate protein using AMPK-3, 2 and -1 antibodies for 3 consecutive immunoprecipitations.

Glycogen content
Muscle glycogen content was measured on 200 g of muscle protein homogenate after acid
hydrolysis as previously described (36).

SDS-PAGE and Western blot analyses

Muscle lysates and homogenates were boiled in Laemmli buffer for 10 min before subjected to
SDS-PAGE and immunoblotting as previously described (30). Quantification of protein
phosphorylation has not been related to protein abundance since expression of all measured proteins
did not change in response to any specified intervention. Small differences in total expression were
observed for some proteins between genotypes however, this did not affect phosphorylation
dynamics or interpretation of data.

Antibodies
Antibodies against phospho-AMPK-Thr172, phospho-ACC/-Ser79/212, Akt2, phospho-Akt-Ser473,
phospho-Akt-Thr308, phospho-TBC1D1-Thr590, phospho-TBC1D4-(Ser318, Ser588, Thr642), phosphoERK1/2-Thr202/Tyr204, and Hexokinase II (HKII) were purchased from Cell Signaling Technology
(Danvers, MA). Antibodies against phospho-TBC1D1-Ser231 and AS160 (TBC1D4) were from
Millipore (Temecula, CA) while antibodies against AMPK2 and GLUT4 were purchased from
Santa Cruz Biotechnology (Dallas, TX) and ThermoFisher Scientific (Waltham, MA), respectively.
ACC protein was determined using HRP-conjugated streptavidin from Dako (Glostrup, Denmark).

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TBC1D1, pyruvate dehydrogenase (PDH), AMPK1 and glycogen synthase (GS) protein as well as
phosphorylation of TBC1D4-Ser711, GS site 2+2a and site 3a+3b were determined using specific
antibodies as previously described (11,36,41). Antibodies used for AMPK activity measurements
were against AMPK2 (Santa Cruz Biotechnology, Dallas, TX), AMPK3 (provided by Professor
D.G. Hardie, University of Dundee, Scotland, UK) and AMPK1 (purchased from GenScript
Jiangning, Nanjing, China).

Statistics
Statistical analyses were performed using SigmaPlot (Version 13.0, SYSTAT, Germany). Two-way
ANOVA with or without repeated measures and paired/unpaired t-tests were used to assess
statistical differences within and between genotypes, where appropriate. A three-way ANOVA was
used to assess differences in total muscle protein abundance between genotypes. The StudentNewman-Keuls test was used for post-hoc testing and all main effects have been indicated by lines.
Correlation analyses were performed by calculating Pearsons product moment correlation
coefficient. Data are expressed as the means SEM unless stated otherwise. Differences were
considered statistically significant at p<0.05.

Results
In situ contraction increases glucose uptake and AMPK signaling in EDL and SOL muscle
During in situ contraction, glucose uptake in EDL and SOL muscle increased similarly in AMPK
WT and mdKO mice (Fig. 1A). Furthermore, in situ contraction decreased muscle glycogen content
(Fig. 1B) and increased Erk1/2 Thr202/Tyr204 phosphorylation (Fig. 1C) to an extent that did not
differ between genotypes. This suggests that the electrical stimulation protocol induced similar
changes in WT and mdKO muscle. In situ contraction markedly increased phosphorylation of

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AMPK Thr172 (Fig. 1D) and downstream targets ACC Ser212 (Fig. 1E), TBC1D1 Ser231 (Fig. 1F)
and TBC1D4 Ser711 (Fig. 1G) in EDL and SOL muscle from WT mice, whereas only minor, if any,
changes were seen in EDL and SOL muscle from mdKO mice. Contraction did not alter total
protein abundance of Erk1/2, AMPK1, AMPK2, ACC, TBC1D1 and TBC1D4 in either EDL or
SOL muscle (Fig. 1H). As expected, EDL and SOL muscle from AMPK mdKO mice showed a
substantial loss of AMPK1 and AMPK2 protein abundance. Identical to previous observations
(36), ACC and TBC1D1 protein abundance was decreased in AMPK mdKO skeletal muscle
compared to WT littermates. Intriguingly, protein abundance of Erk1/2 was increased (~35%,
p<0.01) while TBC1D4 protein abundance was decreased (~20%, p<0.05) in SOL muscle from
mdKO mice compared to WT mice (Fig. 1H).

Prior in situ contraction increases insulin sensitivity in EDL muscle via an AMPK-dependent
mechanism
To test whether the effect of muscle contraction on insulin sensitivity is dependent on AMPK, we
measured submaximal insulin-stimulated glucose uptake ex vivo after in situ contraction. Three
hours after in situ contraction, basal glucose uptake was not significantly different between prior
contracted and rested EDL muscle (Fig. 2A). However, prior in situ contraction increased
submaximal insulin-stimulated glucose uptake in isolated EDL muscle from WT mice but failed to
do so in EDL muscle from AMPK mdKO mice (Fig. 2A). Maximal insulin-stimulated glucose
uptake was similar between prior contracted and rested EDL muscle in both genotypes (Fig. 2A).
The incremental increase in submaximal insulin-stimulated glucose uptake (delta-insulin:
submaximal insulin-stimulated glucose uptake minus basal glucose uptake) was significantly higher
after prior in situ contraction in WT mice only (Fig. 2B). Interestingly, prior in situ contraction did
not increase submaximal insulin-stimulated glucose uptake ex vivo in WT SOL muscle (Fig. 2C and

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D). Based on these results, we performed the subsequent in situ experiments in EDL muscle from
WT and mdKO mice with SOL muscle from WT mice as a negative control.

Prior exercise enhances whole body insulin sensitivity and insulin-stimulated muscle glucose
uptake in WT mice but not in AMPK mdKO mice
To evaluate the involvement of skeletal muscle AMPK in regulating whole body insulin sensitivity
following an acute exercise bout in vivo, we performed intraperitoneal insulin tolerance tests on
AMPK WT and mdKO mice at rest and 1 h after a single bout of acute treadmill exercise. Prior
exercise enhanced the blood glucose lowering response to a submaximal concentration of insulin
(0.3U/kg body weight) (Fig. 2E) and improved insulin tolerance by ~250% in WT mice (Fig. 2G).
In contrast, prior exercise did not induce a greater insulin response to lower blood glucose
concentrations in AMPK mdKO mice (Fig. 2F and G). Furthermore, insulin-stimulated glucose
uptake during the first 40 min of an ITT (0.3U/kg body weight) was significantly improved 1 h after
exercise in m. tibialis anterior from WT mice but not in muscle from AMPK mdKO mice (Fig. 2E
and F).

Total protein abundance in EDL and SOL muscle is not affected by prior muscle contraction
Prior contraction and submaximal insulin did not alter total protein abundance of Akt2, HKII, GS,
ACC, PDH, TBC1D1, TBC1D4, and GLUT4 in EDL muscle within either genotype (Fig. 3A).
Total protein abundance of ACC, TBC1D1, HKII, and PDH was significantly lower (~20-25%;
n=12-13, p<0.05-0.001) in EDL muscle from AMPK mdKO compared to WT mice. In contrast,
Akt2 muscle protein level was ~33% higher (n=12-13, p<0.001) in EDL muscle from mdKO mice
compared to WT mice. No differences in GS, TBC1D4, and GLUT4 muscle protein level was
observed between genotypes. Like in EDL muscle, prior contraction and submaximal insulin did

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not alter total protein abundance of Akt2, TBC1D1, TBC1D4, ACC, and AMPK2 in WT SOL
muscle (Fig. 3B)

Proximal insulin signaling is not enhanced by prior muscle contraction

Previous studies of human, sheep, rat, and mouse investigating muscle insulin sensitivity in the
post-exercise state suggest that the increased ability of insulin to stimulate glucose uptake occurs
independent of enhanced proximal insulin signaling (IR, IRS1, PI3K, and Akt) (57,1518). In
accordance, in the present study prior contraction did also not affect submaximal insulin-induced
phosphorylation of Akt Thr308 and Ser473 in EDL (Fig. 4A and B) or SOL muscle (Fig. 4C and D).

Prior muscle contraction increases site-specific phosphorylation of TBC1D4 in response to

insulin
TBC1D4 is phosphorylated by Akt and AMPK (2426). TBC1D4 has been suggested to regulate
muscle insulin sensitivity, as insulin-stimulated phosphorylation of TBC1D4 is enhanced during
conditions in which muscle displays increased insulin sensitivity following exercise and AICAR
treatment (2730). In the present study, prior contraction of EDL muscle increased the effect of
submaximal insulin stimulation on TBC1D4 Thr649 and Ser711 phosphorylation compared to rested
control muscle from WT mice (Fig. 5A and B). Interestingly, this effect was dependent on AMPK
since insulin-induced phosphorylation of TBC1D4 was similar between rested and prior contracted
EDL muscle from AMPK mdKO mice. Submaximal insulin-stimulated phosphorylation of
TBC1D4 Ser324 and Ser595 was unaffected by prior muscle contraction of EDL muscle (Fig. 5C and
D) suggesting a highly selective interaction between these stimuli. Correlation analyses revealed
that delta-insulin for glucose uptake and delta-insulin for phosphorylation of TBC1D4 Thr649 and
Ser711 in EDL muscle were positively correlated in WT mice (r=0.43-0.65, P < 0.05-0.001; Fig. 5E-

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G). Interestingly, prior contraction did not affect insulin-stimulated phosphorylation of TBC1D4
Thr649 and Ser711 in WT SOL muscle (Fig. 5H and I) in parallel with unchanged insulin sensitivity.

AMPK3-associated activity is increased in EDL muscle 3 hours after in situ contraction

As muscle contraction acutely increases AMPK activity, we investigated whether this effect was
maintained in EDL muscle recovered for 3 hours ex vivo. In muscle from AMPK mdKO mice,
phosphorylation of AMPK Thr172 and downstream target ACC Ser212 was reduced by ~80-90%
compared to WT littermates (Fig. 6A and B). Phosphorylation of AMPK Thr172 and ACC Ser212 had
returned to resting levels 3 hours after contraction. Submaximal insulin did not affect
phosphorylation of AMPK Thr172, but induced a minor increase in ACC Ser212 phosphorylation in
EDL muscle from AMPK mdKO mice (Fig. 6A and B). When measuring AMPK heterotrimer
complex activity 3 hours into recovery, we found that AMPK3-associated activity was increased in
prior contracted WT muscle (P<0.01; Fig. 6C) whereas no significant differences for the remaining
AMPK2- and AMPK1-associated activities were found between rested and prior contracted
muscle (Fig. 6D and E). Phosphorylation of AMPK Thr172 and ACC Ser212 was similar between
prior rested and contracted WT SOL muscle (Fig. 6F and G). Also, no significant differences in
AMPK activity was observed between prior contracted and rested WT SOL muscle (Fig. 6H).
Taken together, this demonstrates that AMPK3-associated activity is increased concomitant with
enhanced muscle insulin sensitivity. To elucidate a possible role of AMPK3 to enhance muscle
insulin sensitivity following contraction, we investigated phosphorylation of TBC1D4 Ser711 in
EDL muscle from AMPK3 KO mice. Interestingly, ex vivo contraction increased phosphorylation
of TBC1D4 Ser711 in WT muscle, but not in muscle from AMPK3 KO mice (Fig. 6I).

Phosphorylation of TBC1D1 does not parallel changes in muscle insulin sensitivity

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Phosphorylation of TBC1D1 has been proposed to regulate muscle glucose uptake in response to
insulin and contraction (42,43). In the present study, submaximal insulin stimulation increased
phosphorylation of TBC1D1 Thr590 in EDL muscle of AMPK mdKO and WT mice, with no
significant differences between rested and prior contracted muscle (Fig. 7A). Furthermore,
phosphorylation of TBC1D1 Ser231 had returned to resting levels 3 hours after contraction and did
not respond to submaximal insulin stimulation (Fig. 7B). Also, phosphorylation of TBC1D1 Ser231
was reduced in EDL muscle from AMPK mdKO mice compared to WT mice (Fig. 7B).
Phosphorylation of TBC1D1 Thr590 and Ser231 in WT SOL muscle (Fig. 7C and D) was similar to
findings in WT EDL muscle, indicating that TBC1D1 is not involved in regulating muscle insulin
sensitivity following contraction.

Increased glycogen synthase (GS) activity does not seem to be necessary for enhanced muscle
insulin sensitivity following contraction
To determine whether GS was secondarily affecting 2-deoxyglucose uptake in skeletal muscle, we
measured GS phosphorylation and activity. In WT mice basal GS activity (%I-form) was similar
between prior rested and contracted EDL muscle (Fig. 8A). In contrast, GS activity was
significantly higher in previously contracted EDL muscle compared with rested muscle in AMPK
mdKO mice. Submaximal insulin stimulation significantly increased GS activity in both rested and
prior contracted EDL muscle independent of genotype (Fig. 8A). Total GS activity was similar
between genotypes and did not change in response to prior contraction or submaximal insulin
stimulation (Fig. 8B). GS activity increases by de-phosphorylation (44). In the present study,
phosphorylation at C-terminal GS residues (3a+3b) decreased similarly in rested and prior
contracted EDL muscle during submaximal insulin stimulation in both genotypes (Fig. 8C). No
significant differences in phosphorylation at N-terminal GS residues (2+2a) were observed in

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response to prior contraction or submaximal insulin stimulation (Fig. 8D). Notably, EDL muscle
from AMPK mdKO mice had a ~40% reduction in GS site 2+2a phosphorylation in line with the
notion that AMPK is a kinase for GS site 2 (45,46). This may explain the higher GS activity
observed in EDL muscle of the AMPK mdKO mouse (Fig. 8A).

Decreased muscle glycogen content is not sufficient to enhance muscle insulin sensitivity after
contraction
In skeletal muscle, insulin-stimulated glucose uptake is suggested to be regulated by glycogen
content per se (47). Three hours after in situ contraction, glycogen content was lower in previously
contracted EDL muscle compared to rested muscle (Fig. 8E). Interestingly, glycogen content was
significantly lower in prior contracted EDL muscle from AMPK mdKO mice compared to EDL
muscle from WT littermates, suggesting that any potential influence of glycogen per se is not a
factor explaining the loss of contraction-induced insulin sensitization in muscle from AMPK mdKO
mice. Notably, glycogen content was similar between prior contracted and rested WT SOL muscle
in parallel with normal insulin sensitivity (Fig. 8F).

Discussion
The present study represents a further contribution to the search of molecular transducers involved
in the insulin sensitizing effect of exercise. Here, we provide evidence to support that AMPK is
necessary for increasing insulin sensitivity to stimulate glucose uptake in EDL muscle following in
situ contraction, as well as enhancing whole body insulin sensitivity and insulin-stimulated muscle
glucose uptake after a single bout of acute exercise. We establish a causal link between a
contraction-regulated signal and the subsequent improvement in muscle insulin sensitivity. Based

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on our findings, we propose that contraction-induced activation of AMPK potentiates the ability of
insulin to increase phosphorylation of TBC1D4 leading to enhanced muscle glucose uptake.
Theoretically, synthesis of new proteins involved in muscle glucose uptake may
mediate improvements in skeletal muscle insulin sensitivity following contraction. However, we
found that greater insulin-stimulated glucose uptake after contraction occurred without an increase
in the abundance of multiple proteins involved in insulin-mediated signaling, as also supported by
findings from others (12,15) and our previous observations in man (5,11,28). We found that the
HKII protein level was decreased in EDL muscle from AMPK mdKO mice compared to WT mice.
However, since maximal insulin-stimulated glucose uptake was similar between genotypes, this
indicates that lower HKII protein abundance in EDL muscle from AMPK mdKO mice is not ratelimiting for the ability of insulin to stimulate glucose uptake following contraction.
Similarly to previous findings in humans and rodents (46,17), the increase in skeletal
muscle insulin sensitivity after contraction was not associated with enhanced proximal insulin
signaling measured at the level of phosphorylated Akt Thr308 and Ser473. This further supports the
notion that the intracellular mechanism responsible for increasing muscle insulin sensitivity after
contraction is located downstream of Akt or involves insulin-regulated parallel pathways
converging with elements regulating glucose transport.
Improved insulin sensitivity after exercise is associated with enhanced translocation of
GLUT4 to the cell surface membrane in skeletal muscle (14). Contraction-induced phosphorylation
of TBC1D1, as well as insulin-induced phosphorylation of TBC1D4 regulates glucose uptake and
GLUT4 translocation in skeletal muscle (26,41,42,48), indicating a role of these proteins in
enhancing skeletal muscle insulin sensitivity after exercise/contraction. Since phosphorylation of
TBC1D1 was similar between genotypes (WT vs. mdKO) and muscle type (EDL vs. SOL), this

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indicates that TBC1D1 is not involved in the insulin-sensitizing effect of prior contraction. This is
supported by several other studies in human and rat (11,17,18,49).
In contrast to TBC1D1, we observed an increased effect of a submaximal dose of
insulin to stimulate phosphorylation of TBC1D4 Thr649 and Ser711 in prior contracted WT EDL
muscle, but not in EDL muscle from AMPK mdKO mice. Furthermore, insulin-stimulated
phosphorylation of TBC1D4 was not enhanced in prior contracted WT SOL muscle. We
hypothesize that the potentiating effect of AMPK activation by prior contraction on insulinstimulated phosphorylation of TBC1D4 Ser711 induces a subsequent increase in TBC1D4 Thr649
phosphorylation which may facilitate the enhanced effect of insulin on glucose uptake. These
observations are supported by positive and significant correlations between TBC1D4 Thr649 and
Ser711 phosphorylation, as well as between glucose uptake and phosphorylation of TBC1D4
Thr649/Ser711 of which Thr649 has previously been reported to be important for muscle glucose
uptake in response to insulin (50). Moreover, these observations are fully in line with our previous
findings in prior AICAR-stimulated EDL muscle (30).
In the present study, contraction-induced phosphorylation of TBC1D4 Ser711 is
dependent on AMPK12 and more specifically on AMPK3. This suggests that the increase in
muscle insulin sensitivity after prior contraction may be mediated through increased AMPK3
activity during and/or after contraction. This idea is supported by observations in the AMPK3scarce SOL muscle (21) in which prior in situ contraction failed to enhance insulin sensitivity,
AMPK3 activity, and phosphorylation of TBC1D4 Ser711. It is also in line with our previous
findings showing that the increased effect of insulin on muscle glucose uptake after prior AICAR
stimulation is dependent on AMPK3 (30). Studies in human and rat have not found evidence to
support increased AMPK activity at time points of enhanced muscle insulin sensitivity after
exercise/contraction (11,18,29). This may be related to measures of AMPK phosphorylation or

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surrogate measures of this (e.g., pACC and pTBC1D1) that potentially conceal differential
regulation among the AMPK heterotrimers (51,52) as also evident in the present study.
The amount of muscle glycogen consumed during an exercise bout may play a role in
regulating post-exercise insulin sensitivity (53). However, we found that the electrical stimulation
protocol decreased glycogen content to similar levels in muscle from both genotypes, indicating that
glycogen depletion per se does not mediate changes in muscle insulin sensitivity as also previously
suggested (54). Skeletal muscle glycogen content and insulin-stimulated glucose uptake display an
inverse relationship (47,55). Thus, glycogen levels at the time of insulin stimulation (rather than
immediately after contraction) may be of importance for muscle insulin sensitivity. However,
immediately before insulin stimulation, muscle glycogen content was lower in prior contracted
mdKO muscle compared to WT muscle indicating that this is not the reason for the lost ability of
prior contraction to enhance muscle insulin sensitivity in AMPK mdKO mice. Since glycogen
content was lower in prior contracted muscle compared to rested muscle in WT mice, we cannot
rule out a functional role of decreased glycogen content for mediating the insulin-sensitizing effect
of prior contraction. This may be supported by observations in prior contracted WT SOL muscle in
which glycogen content had returned to resting levels concomitant with normal insulin sensitivity.
In fact, it may be hypothesized that reduced glycogen levels signal via AMPK to enhance muscle
insulin sensitivity after exercise/contraction. At present we do not possess solid evidence to support
this idea and studies using AICAR (15,30) indicate at least that it is possible to by-pass this
association.
In human and rodent skeletal muscle displaying increased insulin sensitivity after
exercise, GS activity is higher in prior exercised muscle compared to non-exercised muscle
(1,5,9,11). Thus, higher GS activity may be needed for the prior exercised muscle to handle the
increased amount of glucose taken up during insulin stimulation. Because ~30% of 2-deoxyglucose

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taken up by muscle is incorporated into glycogen during insulin stimulation ((56) and unpublished
observations in muscle of mouse), our findings on 2-deoxyglucose uptake may be influenced by
possible dysregulation of GS activity in skeletal muscle of AMPK mdKO mice. However, we found
that in vitro GS activity and phosphorylation were regulated similarly in muscle of AMPK WT and
mdKO mice in response to insulin. In fact, GS activity was elevated in prior rested and prior
exercised muscle from AMPK mdKO mice compared to WT mice. This suggests that elevated GS
activity is not a primary driver for improvements in muscle insulin sensitivity at the level of glucose
uptake.
In conclusion, we provide evidence to support that prior contraction increases insulin
sensitivity in EDL muscle to stimulate glucose uptake by an AMPK-dependent mechanism. Since
the relative distribution of AMPK heterotrimeric complexes in human vastus lateralis greatly
resembles that of mouse EDL muscle (20,21), our findings may be of high relevance for glucose
metabolism in human skeletal muscle as well. Furthermore, we recently found intact regulation of
the AMPK signaling network in skeletal muscle of type 2 diabetic patients (51) and several findings
of enhanced insulin-stimulated phosphorylation of TBC1D4 in prior exercised human muscle
(11,28) support the notion of an AMPK-TBC1D4 signaling axis regulating muscle insulin
sensitivity. Altogether, we provide basic insight to a physiological role of AMPK in skeletal muscle
strengthening the idea of AMPK being a relevant target for physiological and pharmacological
interventions in the prevention and treatment of muscle insulin resistance in various conditions.

Acknowledgments
The authors would like to thank Betina Bolmgren, Irene Bech Nielsen, and Jeppe Kjrgaard Larsen
(Department of Nutrition, Exercise and Sports, Faculty of Science, University of Copenhagen,
Denmark) for their skilled technical assistance.

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Funding
This study was supported by grants from the Danish Council for Independent Research | Medical
Sciences, the research programme (2016) Physical activity and nutrition for improvement of
health funded by the University of Copenhagen, the Lundbeck Foundation, the Novo Nordisk
Foundation, and the Novo Nordisk Foundation Center for Basic Metabolic Research. The Novo
Nordisk Foundation Center for Basic Metabolic Research is an independent Research Center at the
University of Copenhagen that is partially funded by an unrestricted donation from the Novo
Nordisk Foundation (www.metabol.ku.dk).

Duality of interest
The authors have nothing to disclose

Author contributions
R.K. designed and performed the experiments, analysed the data, and wrote the manuscript. N.M.H.
performed the experiments and analysed the data. J.B.B. performed biochemical assays and
analysed the data. M.B., J.R.Z., M.F. and B.V. provided founder mice for the study cohort. J.T.T.
and J.F.P.W. designed the experiments and wrote the manuscript. All authors interpreted the results,
edited and revised the manuscript, and read and approved the final version of the manuscript.
J.F.P.W. is the guarantor of this work and, as such, had full access to all the data in the study and
takes full responsibility for the integrity of the data and the accuracy of the data analysis.

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Figure legends
Figure 1. In situ contraction promotes muscle glucose uptake in WT and AMPK-deficient
mice. 2-deoxyglucose uptake (A), glycogen content (B), pErk1/2 Thr202/Tyr204 (C), pAMPK Thr172
(D), pACC Ser212 (E), pTBC1D1 Ser231 (F) and pTBC1D4 Ser711 (G) in EDL and SOL muscle from
WT (white bars) and AMPK mdKO (black bars) mice immediately after 10 min of in situ
contraction of the lower hindlimb. EDL and SOL muscle were stimulated to contract through the
common peroneal and tibial nerve, respectively. Data were analyzed by two-way repeated measures
ANOVA within each muscle type. A, B, C, D (only SOL) and F: ***p<0.001, **p<0.01, *p<0.05
main effect of contraction. D (only EDL), E and F: treatment genotype interaction (p<0.05),
###p<0.001, ##p<0.01 effect of genotype within treatment; ***p<0.001, **p<0.01, *p<0.05 effect
of contraction within genotype. Representative Western blot images (H). Quantification of protein
phosphorylation has not been related to protein abundance (See Results). Values are means SEM.
For all SOL data, n = 5-6 per group. For all EDL data, n = 3-4 per group except muscle glycogen in
mdKO which has n = 10.

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Figure 2. Improvements in muscle and whole body insulin sensitivity after contraction and
exercise are impaired in AMPK-deficient mice. Glucose uptake (A and C) and delta glucose
uptake (submaximal insulin minus basal) (B and D) in EDL and SOL muscle from AMPK WT and
mdKO mice incubated without or with insulin 2 (SOL) and 3 (EDL) hours after prior in situ
contraction of the lower hindlimb. Blood glucose concentration (% Basal) as well as insulinstimulated muscle glucose uptake (E and F) from AMPK WT and mdKO mice during an insulin
tolerance test (0.3 or 0.4 U/kg) following rest or 1 h after exercise. Absolute blood glucose
concentrations at time point 0 during the 0.3 U/kg ITT in WT mice (Rest: 6.70.2 mmol/L, Prior
exercise: 6.40.2 mmol/L, p=0.36) and in mdKO mice (Rest: 7.40.2 mmol/L, Prior exercise:
6.20.2 mmol/L, p<0.01). Area over the curve calculations (G) were extracted from the 0.3 U/kg
insulin tolerance test in E and F and related to individual rest groups. Data were analyzed by a twoway ANOVA (A, C, E and F) and a t-test (B, D and G) within each genotype (A, C, B, D and G)
and insulin concentration (E and F). A: WT; treatment insulin interaction (p<0.05), ***p<0.001
vs. basal group (0 U/ml) within genotype; ###p<0.001 effect of prior contraction within group;
p<0.001 vs. submaximal group (100 U/ml) within genotype. mdKO; ***p<0.001 vs. basal
group (0 U/ml); p<0.001 vs. submaximal group (100 U/ml). B: Data are extracted from the
raw data in A. #p<0.05 vs. rest within genotype. C: ***p<0.001 main effect of insulin. E: 0.3U/kg;
group time interaction (p<0.05), ***p<0.001, **p<0.01, *p<0.05 effect of group within time;
##p<0.01 effect of prior exercise. G: #p<0.05 vs. rest within genotype. Values are means SEM.
For all SOL data, n = 9-11 per group. For all EDL data, n = 12-13 per group. For insulin tolerance
tests, n=3-4 (0.4 U/kg) and n=6-8 (0.3 U/kg). For in vivo insulin-stimulated muscle glucose uptake,
n=5-8. h, hour.

Figure 3. Insulin and prior contraction do not affect total protein expression. Protein
abundance for Akt2, HKII, GS, ACC, AMPK2, PDH, TBC1D1, TBC1D4 and GLUT4 in EDL

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muscle from AMPK WT and mdKO mice (A) as well as protein abundance for Akt2, TBC1D1,
TBC1D4, ACC and AMPK2 in SOL muscle (B). Data were analyzed by a three-way (A) and a
two-way repeated measure (B) ANOVA. No significant differences were found within each
genotype (A) and SOL muscle (B) in response to prior contraction or submaximal insulin
stimulation. For EDL data, n = 10-12 per group. For SOL data, n = 10-11 per group.

Figure 4. Prior contraction does not affect regulation of Akt by insulin. Phosphorylation of Akt
Thr308 (A and C) and Ser473 (B and D) in EDL muscle from AMPK WT and mdKO mice as well as
WT SOL muscle incubated with or without submaximal insulin 2 (SOL) and 3 (EDL) hours after
prior in situ contraction of the lower hindlimb. Data were analyzed by two-way repeated measures
ANOVA within each genotype (EDL) and muscle (SOL). A-D: ***p<0.001 main effect of insulin.
Quantification of protein phosphorylation has not been related to protein abundance (See Results).
Values are means SEM. For all WT SOL data, n = 11 per group. For all EDL data, n = 11-13 per
group.

Figure 5. Prior contraction increases site-specific phosphorylation of TBC1D4 by insulin.

Phosphorylation of TBC1D4 Thr649 (A and H), Ser711 (B and I), Ser324 (C) and Ser595 (D) in EDL
muscle from AMPK WT and mdKO mice as well as WT SOL muscle incubated with or without
submaximal insulin 2 (SOL) and 3 (EDL) hours after prior in situ contraction of the lower hindlimb.
Data were analyzed by two-way repeated measures ANOVA within each genotype (EDL) and
muscle (SOL). A and B: WT; treatment insulin interaction (p<0.05), ***p<0.001 effect of insulin
within treatment; ##p<0.01 effect of treatment within insulin. mdKO; ***p<0.001 main effect of
insulin. C, D, H and I: ***p<0.001 main effect of insulin. Values are means SEM. Pearson
correlations between delta-insulin (submaximal insulin minus basal) on glucose uptake and
phosphorylation of TBC1D4 Thr649 (E), glucose uptake and phosphorylation of TBC1D4 Ser711 (F)

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as well as phosphorylation of TBC1D4 Thr649 and Ser711 (G). Rest, open symbols; prior contraction,
closed symbols. For all WT SOL data, n = 11 per group. For all EDL data, n = 11-13 per group. rvalues and significance level are indicated in the respective panel. AU, arbitrary units. h, hour.

Figure 6. AMPK3-associated activity is elevated in WT EDL muscle in the post contraction

period. Phosphorylation of AMPK Thr172 (A and F) and ACC Ser212 (B and G) in EDL muscle
from AMPK WT and mdKO mice as well as WT SOL muscle incubated with or without
submaximal insulin 2 (SOL) and 3 (EDL) hours after prior in situ contraction of the lower hindlimb.
AMPK3-associated (C), AMPK21-associated (D) and AMPK1-associated (E) activity in EDL
muscle from AMPK WT and mdKO mice as well as WT SOL muscle (H) 2 (SOL) and 3 (EDL)
hours after prior in situ contraction. Ex vivo contraction-induced phosphorylation of TBC1D4 Ser711
(I) in EDL muscle from AMPK WT and AMPK3 KO mice. Data were analyzed by two-way
repeated measures ANOVA (A, B, F. G and I) and paired t-tests (C-E and H). B: ***p<0.001 main
effect of insulin. C and I: ##p<0.01, #p<0.05 vs. rest within genotype. Quantification of protein
phosphorylation has not been related to protein abundance (See Results). Values are means SEM.
For WT SOL data, n = 10-11 per group in panel F and G and n = 7-8 in panel H. For EDL data, n =
12-13 per group in panel A and B and n = 7-10 per group in panel C-E and I.

Figure 7. Prior contraction does not affect regulation of TBC1D1 by insulin. Phosphorylation
of TBC1D1 Thr590 (A and C) and Ser231 (B and D) in EDL muscle from AMPK WT and mdKO
mice as well as WT SOL muscle incubated with or without submaximal insulin 2 (SOL) and 3
(EDL) hours after prior in situ contraction of the lower hindlimb. Data were analyzed by two-way
repeated measures ANOVA within each genotype (EDL) and muscle (SOL). A and C: ***p<0.001
main effect of insulin. Quantification of protein phosphorylation has not been related to protein

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abundance (See Results). Values are means SEM. For all WT SOL data, n = 11 per group. For all
EDL data, n = 12-13 per group.

Figure 8. Glycogen synthase activity does not drive improvements in muscle insulin sensitivity
after prior contraction. Glycogen synthase (GS) activity expressed as %I-form (A) and total (B)
as well as phosphorylation of GS site 3a+3b (C) and 2+2a (D) in EDL muscle from AMPK WT and
mdKO mice incubated with or without submaximal insulin 3 hours after prior in situ contraction of
the lower hindlimb. Glycogen content (E and F) in EDL muscle from AMPK WT and mdKO mice
as well as WT SOL muscle 2 (SOL) and 3 (EDL) hours into recovery from prior in situ contraction.
Data were analyzed by two-way repeated measures ANOVA within each genotype (A-D) and
between genotypes (E) as well as a paired t-test (F). A and C: ***p<0.001 main effect of insulin
within genotype. E: treatment genotype interaction (p<0.05), ###p<0.001, ##p<0.01 effect of
treatment within genotype; p<0.05 effect of genotype within treatment. Values are means
SEM. For WT SOL data, n = 8 per group. For EDL data, n = 12-13 per group in panel A-D and n =
10 per group in panel E.

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Figure
1

Glucose uptake

450
400
350
300
250
200
150
100
50
0

EDL

***

Rest

WT
mdKO

SOL

***

Contraction

Rest

EDL

WT
mdKO

SOL

100

60
40
20

Rest

EDL

WT
mdKO

SOL

Relative units

###

Rest

Contraction

Main effect of genotype (p<0.001)

2
##

Rest

Contraction

Rest

Contraction

pTBC1D1 Ser231
5

EDL

Main effect of genotype (p<0.01)

**

3
2
1

###

Rest

***

Contraction

EDL

SOL

WT
mdKO

###

*
##
Relative units

pTBC1D4 Ser711

**
0

Rest

Contraction

WT
mdKO

SOL

Rest

Contraction

Rest

Rest

Contraction

Rest#

Contraction
SOL#

EDL#

WT
mdKO

SOL

F
Relative units

***
##

Contraction

Main effect of genotype (p<0.01)

10
5

***
###

15

Rest

***
##

20

Contraction

EDL

Contraction

pACC Ser212

25

Relatvie units

Contraction

Rest

pAMPK Thr172

Rest

p=0.05

80

Relative units

***

WT
mdKO

SOL

***

EDL

120

***
6

Glycogen

140

Contraction

pErk1/2 Thr202/Tyr204
8

Glycosyl units [pmol / g protein]

2-deoxyglucose uptake
ng glucose / mg tissue / min

Page 30 of 37

Rest#

Contrac2on#

Contrac2on#

WT# mdKO# WT# mdKO#

WT# mdKO# WT# mdKO#

pAMPK#Thr172#

pAMPK#Thr172#

pACC#Ser212#

pACC#Ser212#

pTBC1D1#Ser231#

pTBC1D1#Ser231#

pTBC1D4#Ser711#

pTBC1D4#Ser711#

pErk1/2#Thr202/Tyr204#

pErk1/2#Thr202/Tyr204#

ACC#

ACC#

TBC1D1#

TBC1D1#

TBC1D4#

TBC1D4#

Erk1/2#

Erk1/2#

AMPK1#

AMPK1#

AMPK2#

AMPK2#

Beta?ac2n#

GAPDH#

Figure
Diabetes2

Page 31 of 37

EDL

50

Rest
Prior contraction

40
###

30

***

***
20
10

20
15
10
5
0

100

10.000

100

WT

40
30
20
10
0

100

WT

100

0.3U/kg

***

Rest
Prior Exercise

2-deoxyglucose uptake
ng glucose / mg tissue / min

80
70
60

20

40

60

80

100

20
15
10

##

5
0

TA

120

Time after insulin injection [min]

Rest 0.3 U/kg
Rest 0.4 U/kg

G
Relative to Rest [%]

600

400
300
200
100
0

WT

15
10
5

WT

mdKO

mdKO

EDL

mdKO

110
0.3U/kg

100

Rest
Prior Exercise

90
80
70
60
50
10
0

20

40

60

80

100

120

Time after insulin injection [min]

Prior exercise 0.3 U/kg

Prior exercise 0.4 U/kg

Area over the curve

0-40 min

500

20

90

50
10
0

25

p=0.1

**

30

Insulin (U/ml)

WT

100

Rest
Prior contraction

mdKO

E
110

35

2-deoxyglucose uptake
[mol / g protein / h]

2-deoxyglucose uptake
[mol / g protein / h]

Rest
Prior contraction

***

***

mdKO

Delta Submaximal Insulin

Glucose uptake in SOL

SOL

50

WT

Insulin (U/ml)

mdKO

Blood glucose [% Basal]

10.000

Blood glucose [% Basal]

Rest
Prior contraction

2-deoxyglucose uptake
ng glucose / mg tissue / min

2-deoxyglucose uptake
[mol / g protein / h]

***

Delta Submaximal Insulin

Glucose uptake in EDL

B
2-deoxyglucose uptake
[mol / g protein / h]

Rest
Prior contraction

Rest 0.3 U/kg

Prior exercise 0.3 U/kg

Rest 0.4 U/kg

Prior exercise 0.4 U/kg

25
20
15
10
5
0

TA

EDL

Figure
Diabetes 3

A
Prior contraction
Submaximal insulin

Page 32 of 37

EDL
-

WT
+
+

+
+

mdKO
+
- +

WT"SOL"
Basal"

+
+

Prior"contracBon"

Akt2
HKII

!"

+"

Insulin"
!"

+"
Akt2"
TBC1D1"

GS

TBC1D4"

ACC

ACC"

PDH

AMPK2"

TBC1D1
TBC1D4
GLUT4

AMPK1
AMPK2
GAPDH

GAPDH"

Figure
Diabetes4

Page 33 of 37

A
10

Rest
Prior contraction

***

***

6
4

12

100

100

6
4
0

D
Relative units

***

4
2

100

Insulin (U/ml)

100

100

Insulin (U/ml)

mdKO

WT SOL pAkt Ser473

8

Rest
Prior contraction

WT

mdKO

Relative units

Insulin (U/ml)

WT SOL pAkt Thr308

***

10

WT

Rest
Prior contraction

***

14

2
0

EDL pAkt Ser473

16

Relative units

Relative units

EDL pAkt Thr308

Rest
Prior contraction

***

6
4
2
0

100

Insulin (U/ml)

Figure
Diabetes5

EDL pTBC1D4 Thr649

Rest
Prior contraction

4
###

1
0

100

WT

100

***

***

mdKO

100

WT

EDL pTBC1D4 Ser324

Rest
Prior contraction

D
Relative units

Insulin (U/ml)

mdKO

EDL pTBC1D4 Ser595

Rest
Prior contraction

***

***

100

***

***

Rest
Prior contraction

##

***

Insulin (U/ml)

Relative units

Relative units

Relative units

***

EDL pTBC1D4 Ser711

***

***

Page 34 of 37

3
2
1

100

WT

Rest

100

Prior contraction

Rest

Prior contraction

10

-0.5

r=0.47
p<0.05

10

0.0

0.5

1.0

WT SOL pTBC1D4 Thr649

Rest

Prior contraction

I
Relative units

***

1.5

2.0

2.5

4
3
2

-0.5

Rest

Insulin (U/ml)

100

Prior contraction

r=0.65
p<0.001

0.0

0.5

1.0

1.5

2.0

pTBC1D4 Ser711 [AU]

WT SOL pTBC1D4 Ser711

Rest
Prior contraction

***

1
0

Insulin (U/ml)

pTBC1D4 Ser711 [AU]

pTBC1D4 Thr649 [AU]

H7

20

100

mdKO

pTBC1D4 Thr649 [AU]

r=0.43
p<0.05

Glucose uptake
[mol / g protein / h]

20

WT

30

Relative units

Glucose uptake
[mol / g protein / h]

Insulin (U/ml)

mdKO

30

100

Insulin (U/ml)

100

Figure
6
Diabetes

Page 35 of 37

EDL pAMPK Thr172

1.5
Rest
Prior contraction

1.0

0.5

Relative units

Relative units

1.5

EDL pACC Ser212

Rest
Prior contraction

1.0

0.5

***
100

WT

1.0

##

Rest
Prior contraction

0.4
0.2

WT

0.8
0.6
0.4
0.2
0.0

WT

4
2

WT

mdKO

WT Soleus
pAMPK Thr172

1.0
0.5

WT Soleus
pACC Ser212

G
2.0

Rest
Prior contraction

1.5

0.0

mdKO

Rest
Prior contraction

2.0

Rest
Prior contraction

Rest
Prior contraction

1.5
1.0
0.5
0.0

100

Rest
Prior contraction

p=0.28

2
1
p=0.42

AMPK3associated

EDL pTBC1D4 Ser711

2.0

p=0.07

#
Relative units

[ pmol min-1 mg-1 ]

AMPK activity in WT SOL muscle

2 h after in situ contraction

AMPK1associated

Rest
Contraction

1.5
AMPK"WT"

!"

1.0

+"

AMPK3"KO"
!"

+"

Contrac=on"
pTBC1D4"Ser711"
TBC1D4"

0.5
0.0

AMPK21associated

100

Insulin (U/ml)

Insulin (U/ml)

Insulin (U/ml)

mdKO

p=0.13

F
Relative units

1.0

100

AMPK1-associated activity in EDL

muscle 3 h after in situ contraction
p=0.10

AMPK21-associated activity in EDL

muscle 3 h after in situ contraction

mdKO

1.2

100

WT

0.6

mdKO

0.8

0.0

[ pmol min-1 mg-1 ]

0.0
Insulin (U/ml)

AMPK3-associated activity in EDL

muscle 3 h after in situ contraction

C
[ pmol min-1 mg-1 ]

100

Relative units

[ pmol min-1 mg-1 ]

0.0

pACC"Ser212"
ACC"

AMPK WT AMPK3 KO

Figure
Diabetes7

EDL pTBC1D1 Thr590

2.5

***

Relative units

Relative units

***

1.5
1.0
0.5
0.0

100

WT

100

0.5

Insulin (U/ml)

100

100

100

WT Soleus pTBC1D1 Ser231

Rest
Prior contraction

1.5
1.0
0.5
0.0

Insulin (U/ml)

mdKO

2.0

Relative units

Relative units

1.0

WT

***

0.5
0.0

Rest
Prior contraction

1.5

0.0

1.0

Insulin (U/ml)

WT Soleus pTBC1D1 Thr590

2.0

Rest
Prior contraction

1.5

mdKO

2.5

EDL pTBC1D1 Ser231

2.0

Rest
Prior contraction

2.0

Page 36 of 37

Insulin (U/ml)

100

Figure
Diabetes8

Page 37 of 37

A
40

Rest
Prior contraction
Main effect of prior contraction
within mdKO (p<0.01)

30

***

20
10
0

100

WT

100

Relative units

***

100

100

Insulin (U/ml)

200

##

###

Rest

Prior contraction

100

Insulin (U/ml)

mdKO

Rest
Prior contraction

1.0

0.5

0.0

100

WT

100

Insulin (U/ml)

mdKO

Glycogen content in WT SOL muscle

2 h after in situ contraction

F
WT
mdKO

50
0

pGS 2+2a

mdKO

Glycogen content in EDL muscle

3 h after in situ contraction

100

100

1.5

Rest
Prior contraction

0.5

150

WT

Glycosyl units [pmol / g protein]

Relative units
Glycosyl units [pmol / g protein]

1.0

***

WT

10

mdKO

1.5

Rest
Prior contraction

15

Insulin (U/ml)

pGS 3a+3b

0.0

GS total activity

20

***

GS activity
[mmol / mg protein / min]

50

GS activity: I-form (%)

GS I-form

150

100

50

Rest

Prior contraction