Page 1 of 37 Diabetes
Enhanced muscle insulin sensitivity after contraction/exercise is mediated by AMPK
Rasmus Kjøbsted1,2, Nanna Munk Hansen1, Jesper B. Birk1, Marc Foretz3,4,5, Benoit Viollet3,4,5, Marie Björnholm6, Juleen R. Zierath2,6, Jonas T. Treebak2, Jørgen 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 CNRS, UMR8104, Paris, France 5 Université Paris Descartes, Sorbonne Paris Cité, Paris, France 6Integrative 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: Jørgen 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: [email protected]
Diabetes Publish Ahead of Print, published online October 26, 2016 Diabetes Page 2 of 37
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 carboxamide ribonucleotide (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 AMPKα1α2 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 insulin stimulated 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.
2 Page 3 of 37 Diabetes
Introduction
Skeletal muscle from both human, sheep, dog, and rodents demonstrates increased insulin
stimulated glucose uptake in the period after a single bout of exercise (1–10). This phenomenon is
observed in both normal and insulin resistant muscle (11–13) 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 α and regulatory β and γ subunits of which several isoforms exist (α1, α2, β1, β2, γ1, γ2
and γ3) (19). In human and mouse skeletal muscle, 3 (α2β2γ1, α2β2γ3 and α1β2γ1) and 5 (α2β2γ1,
α2β2γ3, α2β1γ1, α1β2γ1 and α1β1γ1) heterotrimeric combinations have been found, respectively
(20,21). Interestingly, mouse skeletal muscle contains two β1 associated complexes that are not
found in human skeletal muscle. Furthermore, in mouse EDL and human vastus lateralis muscle,
the α2β2γ3 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).
3 Diabetes Page 4 of 37
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 AICAR induced 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
(11,17,18,27–29).
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 AICAR and exercise/contraction induced biological responses are equally dependent on AMPK activation.
Since the first proposal of an insulin sensitizing effect of prior exercise by Bergström 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
4 Page 5 of 37 Diabetes
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 2934
01037; #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 AMPKα1α2 muscle specific double KO and whole body AMPKγ3 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
5 Diabetes Page 6 of 37
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 Krebs
Ringer 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 30°C. 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]2 deoxyglucose (0.028 MBq/mL), 7 mmol/L [14C]mannitol (0.0083 MBq/mL) and 2 mmol/L
6 Page 7 of 37 Diabetes
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 µl / g body weight) containing 0.1 mM [3H]2
deoxyglucose (1,78 MBq) 1 h after rest and exercise. Immediately before, as well as 20 and 40 min
7 Diabetes Page 8 of 37
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 AMPKγ3 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 µL ice cold homogenization buffer (30) and rotated end over end 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 µg 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
8 Page 9 of 37 Diabetes
Heterotrimer specific AMPK activity in mouse skeletal muscle was determined as previously
described (30). AMPK activity was measured on 300 µg of muscle lysate protein using AMPK γ3,
α2 and α1 antibodies for 3 consecutive immunoprecipitations.
Glycogen content
Muscle glycogen content was measured on 200 µg 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 ACCα/β Ser79/212, Akt2, phospho Akt Ser473,
phospho Akt Thr308, phospho TBC1D1 Thr590, phospho TBC1D4 (Ser318, Ser588, Thr642), phospho
ERK1/2 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 AMPKα2 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).
9 Diabetes Page 10 of 37
TBC1D1, pyruvate dehydrogenase (PDH), AMPKα1 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 AMPKα2 (Santa Cruz Biotechnology, Dallas, TX), AMPKγ3 (provided by Professor
D.G. Hardie, University of Dundee, Scotland, UK) and AMPKα1 (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 Student
Newman Keuls test was used for post hoc testing and all main effects have been indicated by lines.
Correlation analyses were performed by calculating Pearson’s 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
10 Page 11 of 37 Diabetes
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, AMPKα1, AMPKα2, 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 AMPKα1 and AMPKα2 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
11 Diabetes Page 12 of 37
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
12 Page 13 of 37 Diabetes
not alter total protein abundance of Akt2, TBC1D1, TBC1D4, ACC, and AMPKα2 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) (5–7,15–18). 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 (24–26). 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 (27–30). 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
13 Diabetes Page 14 of 37
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.
AMPKγ3 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 AMPKγ3 associated activity was increased in prior contracted WT muscle (P<0.01; Fig. 6C) whereas no significant differences for the remaining
AMPKα2 and AMPKα1 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 AMPKγ3 associated activity is increased concomitant with enhanced muscle insulin sensitivity. To elucidate a possible role of AMPKγ3 to enhance muscle insulin sensitivity following contraction, we investigated phosphorylation of TBC1D4 Ser711 in
EDL muscle from AMPKγ3 KO mice. Interestingly, ex vivo contraction increased phosphorylation of TBC1D4 Ser711 in WT muscle, but not in muscle from AMPKγ3 KO mice (Fig. 6I).
Phosphorylation of TBC1D1 does not parallel changes in muscle insulin sensitivity
14 Page 15 of 37 Diabetes
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
15 Diabetes Page 16 of 37
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
16 Page 17 of 37 Diabetes
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 rate
limiting for the ability of insulin to stimulate glucose uptake following contraction.
Similarly to previous findings in humans and rodents (4–6,17), 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
17 Diabetes Page 18 of 37
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 insulin stimulated 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 AMPKα1α2 and more specifically on AMPKγ3. This suggests that the increase in muscle insulin sensitivity after prior contraction may be mediated through increased AMPKγ3 activity during and/or after contraction. This idea is supported by observations in the AMPKγ3 scarce SOL muscle (21) in which prior in situ contraction failed to enhance insulin sensitivity,
AMPKγ3 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 AMPKγ3 (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
18 Page 19 of 37 Diabetes
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
19 Diabetes Page 20 of 37
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 Kjærgaard Larsen
(Department of Nutrition, Exercise and Sports, Faculty of Science, University of Copenhagen,
Denmark) for their skilled technical assistance.
20 Page 21 of 37 Diabetes
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.
21 Diabetes Page 22 of 37
References
1. Richter EA, Garetto LP, Goodman MN, Ruderman NB. Muscle glucose metabolism following exercise in the rat: increased sensitivity to insulin. J Clin Invest. 1982;69(4):785–93. 2. Richter EA, Mikines KJ, Galbo H, Kiens B. Effect of exercise on insulin action in human skeletal muscle. J Appl Physiol. 1989;66(2):876–85. 3. Cartee GD, Holloszy JO. Exercise increases susceptibility of muscle glucose transport to activation by various stimuli. Am J Physiol. 1990;258(2 Pt 1):E390–3. 4. Wojtaszewski JFP, Hansen BF, Kiens B, Richter EA. Insulin Signaling in Human Skeletal Muscle: Time Course and Effect of Exercise. Diabetes. 1997;46(11):1775–81. 5. Wojtaszewski JFP, Hansen BF, Gade J, Kiens B, Markuns JF, Goodyear LJ, et al. Insulin signaling and insulin sensitivity after exercise in human skeletal muscle. Diabetes. 2000;49(3):325–31. 6. Hamada T, Arias EB, Cartee GD. Increased submaximal insulin-stimulated glucose uptake in mouse skeletal muscle after treadmill exercise. J Appl Physiol. 2006;101(5):1368–76. 7. McConell GK, Kaur G, Falcão-Tebas F, Hong YH, Gatford KL. Acute exercise increases insulin sensitivity in adult sheep: a new preclinical model. Am J Physiol - Regul Integr Comp Physiol. 2015;308(6):R500–6. 8. Pencek RR, James F, Lacy DB, Jabbour K, Williams PE, Fueger PT, et al. Interaction of insulin and prior exercise in control of hepatic metabolism of a glucose load. Diabetes. 2003;52(8):1897–903. 9. Bogardus C, Thuillez P, Ravussin E, Vasquez B, Narimiga M, Azhar S. Effect of muscle glycogen depletion on in vivo insulin action in man. J Clin Invest. 1983;72(5):1605–10. 10. Mikines KJ, Sonne B, Farrell PA, Tronier B, Galbo H. Effect of physical exercise on sensitivity and responsiveness to insulin in humans. Am J Physiol. 1988;254(3 Pt 1):E248–59. 11. Pehmøller C, Brandt N, Birk JB, Høeg LD, Sjøberg K, Goodyear LJ, et al. Exercise Alleviates Lipid- Induced Insulin Resistance in Human Skeletal Muscle–Signaling Interaction at the Level of TBC1 Domain Family Member 4. Diabetes. 2012;61(11):2743–52. 12. Castorena CM, Arias EB, Sharma N, Cartee GD. Postexercise improvement in insulin-stimulated glucose uptake occurs concomitant with greater AS160 phosphorylation in muscle from normal and insulin-resistant rats. Diabetes. 2014;63(7):2297–308. 13. Devlin JT, Hirshman M, Horton ED, Horton ES. Enhanced peripheral and splanchnic insulin sensitivity in NIDDM men after single bout of exercise. Diabetes. 1987;36(4):434–9. 14. Hansen PA, Nolte LA, Chen MM, Holloszy JO. Increased GLUT-4 translocation mediates enhanced insulin sensitivity of muscle glucose transport after exercise. J Appl Physiol. 1998;85(4):1218–22. 15. Fisher JS, Gao J, Han D-H, Holloszy JO, Nolte LA. Activation of AMP kinase enhances sensitivity of muscle glucose transport to insulin. Am J Physiol Endocrinol Metab. 2002;282(1):E18–23. 16. Bonen A, Tan MH, Watson-Wright WM. Effects of exercise on insulin binding and glucose metabolism in muscle. Can J Physiol Pharmacol. 1984;62(12):1500–4. 17. Funai K, Schweitzer GG, Sharma N, Kanzaki M, Cartee GD. Increased AS160 phosphorylation, but not TBC1D1 phosphorylation, with increased postexercise insulin sensitivity in rat skeletal muscle. Am J Physiol Endocrinol Metab. 2009;297(1):E242–51. 18. Funai K, Schweitzer GG, Castorena CM, Kanzaki M, Cartee GD. In vivo exercise followed by in vitro contraction additively elevates subsequent insulin-stimulated glucose transport by rat skeletal
22 Page 23 of 37 Diabetes
muscle. Am J Physiol Endocrinol Metab. 2010;298(5):E999–1010. 19. Hardie DG, Scott JW, Pan DA, Hudson ER. Management of cellular energy by the AMP-activated protein kinase system. FEBS Lett. 2003;546(1):113–20. 20. Wojtaszewski JFP, Birk JB, Frøsig C, Holten M, Pilegaard H, Dela F. 5’AMP activated protein kinase expression in human skeletal muscle: effects of strength training and type 2 diabetes. J Physiol. 2005;564(Pt 2):563–73. 21. Treebak JT, Birk JB, Hansen BF, Olsen GS, Wojtaszewski JFP. A-769662 activates AMPK beta1- containing complexes but induces glucose uptake through a PI3-kinase-dependent pathway in mouse skeletal muscle. Am J Physiol Cell Physiol. 2009;297(4):C1041–52. 22. Kahn BB, Alquier T, Carling D, Hardie DG. AMP-activated protein kinase: Ancient energy gauge provides clues to modern understanding of metabolism. Cell Metab. 2005;1(1):15–25. 23. Hardie DG, Ross FA., Hawley SA. AMPK: a nutrient and energy sensor that maintains energy homeostasis. Nat Rev Mol Cell Biol. 2012;13(4):251–62. 24. Kramer HF, Witczak CA, Fujii N, Jessen N, Taylor EB, Arnolds DE, et al. Distinct signals regulate AS160 phosphorylation in response to insulin, AICAR, and contraction in mouse skeletal muscle. Diabetes. 2006;55(7):2067–76. 25. Treebak JT, Taylor EB, Witczak CA, An D, Toyoda T, Koh H-J, et al. Identification of a novel phosphorylation site on TBC1D4 regulated by AMP-activated protein kinase in skeletal muscle. Am J Physiol Cell Physiol. 2010;298(2):C377–85. 26. Kramer HF, Witczak CA, Taylor EB, Fujii N, Hirshman MF, Goodyear LJ. AS160 regulates insulin- and contraction-stimulated glucose uptake in mouse skeletal muscle. J Biol Chem. 2006;281(42):31478– 85. 27. Arias EB, Kim J, Funai K, Cartee GD. Prior exercise increases phosphorylation of Akt substrate of 160 kDa (AS160) in rat skeletal muscle. Am J Physiol Endocrinol Metab. 2007;292(4):E1191–200. 28. Treebak JT, Frøsig C, Pehmøller C, Chen S, Maarbjerg SJ, Brandt N, et al. Potential role of TBC1D4 in enhanced post-exercise insulin action in human skeletal muscle. Diabetologia. 2009;52(5):891–900. 29. Schweitzer GG, Arias EB, Cartee GD. Sustained postexercise increases in AS160 Thr642 and Ser588 phosphorylation in skeletal muscle without sustained increases in kinase phosphorylation. J Appl Physiol. 2012;113(12):1852–61. 30. Kjøbsted R, Treebak JT, Fentz J, Lantier L, Viollet B, Birk JB, et al. Prior AICAR stimulation increases insulin sensitivity in mouse skeletal muscle in an AMPK-dependent manner. Diabetes. 2015;64(6):2042–55. 31. Jørgensen SB, Viollet B, Andreelli F, Frøsig C, Birk JB, Schjerling P, et al. Knockout of the α2 but not α1 5'-AMP-activated protein kinase isoform abolishes 5-aminoimidazole-4-carboxamide-1-β-4- ribofuranoside- but not contraction-induced glucose uptake in skeletal muscle. J Biol Chem. 2004;279(2):1070-79. 32. Jørgensen SB, Wojtaszewski JFP, Viollet B, Andreelli F, Birk JB, Hellsten Y, et al. Effects of α-AMPK knockout on exercise-induced gene activation in mouse skeletal muscle. FASEB J. 2005;19(9):1146-8. 33. Jørgensen SB, Treebak JT, Viollet B, Schjerling P, Vaulont S, Wojtaszewski JFP, et al. Role of AMPKα2 in basal, training-, and AICAR-induced GLUT4, hexokinase II, and mitochondrial protein expression in mouse muscle. Am J Physiol Endocrinol Metab. 2007;292(1):E331-9. 34. Jeppesen J, Albers PH, Rose AJ, Birk JB, Schjerling P, Dzamko N, et al. Contraction-induced skeletal muscle FAT/CD36 trafficking and FA uptake is AMPK independent. J Lipid Res. 2011;52(4):699-711.
23 Diabetes Page 24 of 37
35. Lantier L, Fentz J, Mounier R, Leclerc J, Treebak JT, Pehmøller C, et al. AMPK controls exercise endurance, mitochondrial oxidative capacity, and skeletal muscle integrity. FASEB J. 2014;28(7):3211–24. 36. Fentz J, Kjøbsted R, Birk JB, Jordy AB, Jeppesen J, Thorsen K, et al. AMPKα is critical for enhancing skeletal muscle fatty acid utilization during in vivo exercise in mice. FASEB J. 2015;29(5):1725–38. 37. Bergström J, Hultman E. Muscle glycogen synthesis after exercise: an enhancing factor localized to the muscle cells in man. Nature. 1966;210(5033):309-10. 38. Barnes BR, Marklund S, Steiler TL, Walter M, Hjälm G, Amarger V, et al. The 5′-AMP-activated protein kinase γ3 isoform has a key role in carbohydrate and lipid metabolism in glycolytic skeletal muscle. J Biol Chem. 2004;279(37):38441–7. 39. Ferré P, Leturque A, Burnol AF, Penicaud L, Girard J. A method to quantify glucose utilization in vivo in skeletal muscle and white adipose tissue of the anaesthetized rat. Biochem J. 1985;228(1):103– 10. 40. Treebak JT, Glund S, Deshmukh A, Klein DK, Long YC, Jensen TE, et al. AMPK-mediated AS160 phosphorylation in skeletal muscle is dependent on AMPK catalytic and regulatory subunits. Diabetes. 2006;55(7):2051-8. 41. Pedersen AJT, Hingst JR, Friedrichsen M, Kristensen JM, Højlund K, Wojtaszewski JFP. Dysregulation of muscle glycogen synthase in recovery from exercise in type 2 diabetes. Diabetologia. 2015;(2015):1569–78. 42. Vichaiwong K, Purohit S, An D, Toyoda T, Jessen N, Hirshman MF, et al. Contraction regulates site- specific phosphorylation of TBC1D1 in skeletal muscle. Biochem J. 2010;431(2):311–20. 43. An D, Toyoda T, Taylor EB, Yu H, Fujii N, Hirshman MF, et al. TBC1D1 regulates insulin- and contraction-induced glucose transport in mouse skeletal muscle. Diabetes. 2010;59(6):1358–65. 44. Skurat AV, Dietrich AD, Roach PJ. Glycogen synthase sensitivity to insulin and glucose-6-phosphate is mediated by both NH2- and COOH-terminal phosphorylation sites. Diabetes. 2000;49(7):1096–100. 45. Jørgensen SB, Nielsen JN, Birk JB, Olsen GS, Viollet B, Andreelli F, et al. The α2-5'AMP-activated protein kinase is a site 2 glycogen synthase kinase in skeletal muscle and is responsive to glucose loading. Diabetes. 2004;53(12):3074–81. 46. Carling D, Hardie DG. The substrate and sequence specificity of the AMP-activated protein kinase. Phosphorylation of glycogen synthase and phosphorylase kinase. Biochim Biophys Acta. 1989;1012(1):81–6. 47. Jensen J, Aslesen R, Ivy JL, Brørs O. Role of glycogen concentration and epinephrine on glucose uptake in rat epitrochlearis muscle. Am J Physiol. 1997;272(4 Pt 1):E649–55. 48. Thong FSL, Bilan PJ, Klip A. The Rab GTPase-activating protein AS160 integrates Akt, protein kinase C, and AMP-activated protein kinase signals regulating GLUT4 traffic. Diabetes. 2007;56(2):414–23. 49. Iwabe M, Kawamoto E, Koshinaka K, Kawanaka K. Increased postexercise insulin sensitivity is accompanied by increased AS160 phosphorylation in slow-twitch soleus muscle. Physiol Rep. 2014;2(12):1–9. 50. Chen S, Wasserman DH, MacKintosh C, Sakamoto K. Mice with AS160/TBC1D4-Thr649Ala knockin mutation are glucose intolerant with reduced insulin sensitivity and altered GLUT4 trafficking. Cell Metab. 2011;13(1):68–79. 51. Kjøbsted R, Pedersen AJT, Hingst JR, Sabaratnam R, Birk JB, Kristensen JM, et al. Intact regulation of the AMPK signaling network in response to exercise and insulin in skeletal muscle of male patients with type 2 diabetes: Illumination of AMPK activation in recovery from exercise. Diabetes.
24 Page 25 of 37 Diabetes
2016;65(5):1219-30 52. Birk JB, Wojtaszewski JFP. Predominant α2/β2/γ3 AMPK activation during exercise in human skeletal muscle. J Physiol. 2006;577(Pt 3):1021–32. 53. Richter EA, Derave W, Wojtaszewski JFP. Glucose, exercise and insulin: Emerging concepts. J Physiol. 2001;535(2):313–22. 54. Kim J, Solis RS, Arias EB, Cartee GD. Postcontraction insulin sensitivity: relationship with contraction protocol, glycogen concentration, and 5’ AMP-activated protein kinase phosphorylation. J Appl Physiol. 2004;96(2):575–83. 55. Derave W, Hansen BF, Lund S, Kristiansen S, Richter EA. Muscle glycogen content affects insulin- stimulated glucose transport and protein kinase B activity. Am J Physiol Endocrinol Metab. 2000;279(5):E947–55. 56. Virkamäki A, Rissanen E, Hämäläinen S, Utriainen T, Yki-Järvinen H. Incorporation of [3-3H]glucose and 2-[1-14C]deoxyglucose into glycogen in heart and skeletal muscle in vivo: implications for the quantitation of tissue glucose uptake. Diabetes. 1997;46(7):1106–10.
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.
25 Diabetes Page 26 of 37
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 insulin stimulated 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: 6.7±0.2 mmol/L, Prior exercise: 6.4±0.2 mmol/L, p=0.36) and in mdKO mice (Rest: 7.4±0.2 mmol/L, Prior exercise:
6.2±0.2 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 two way 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 µU/ml) within genotype; ###p<0.001 effect of prior contraction within group;
§§§p<0.001 vs. submaximal group (100 µU/ml) within genotype. mdKO; ***p<0.001 vs. basal group (0 µU/ml); §§§p<0.001 vs. submaximal group (100 µU/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, AMPKα2, PDH, TBC1D1, TBC1D4 and GLUT4 in EDL
26 Page 27 of 37 Diabetes
muscle from AMPK WT and mdKO mice (A) as well as protein abundance for Akt2, TBC1D1,
TBC1D4, ACC and AMPKα2 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)
27 Diabetes Page 28 of 37
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. r values and significance level are indicated in the respective panel. AU, arbitrary units. h, hour.
Figure 6. AMPKγ3 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.
AMPKγ3 associated (C), AMPKα2βγ1 associated (D) and AMPKα1 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 AMPKγ3 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
28 Page 29 of 37 Diabetes
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.
29 FigureDiabetes 1 Page 30 of 37
A Glucose uptake B Glycogen 450 WT 140 WT EDL SOL mdKO EDL SOL mdKO 400 120 p=0.05 350 *** *** 100 300 *** 250 80 200 60 150 40 100 2-deoxyglucose uptake 2-deoxyglucose 20
ng glucose / mg tissue / min / tissue mg / glucose ng 50
0 Glycosyl units [pmol / µg protein] 0 Rest Contraction Rest Contraction Rest Contraction Rest Contraction
C pErk1/2 Thr202/Tyr204 D pAMPK Thr172 WT WT 8 EDL SOL mdKO 6 EDL SOL mdKO *** *** Main effect of genotype (p<0.001) 5 ### 6 *** 4
4 3 * Relative units Relative Relative units Relative 2 2 ## 1
0 0 Rest Contraction Rest Contraction Rest Contraction Rest Contraction
E pACC Ser212 F pTBC1D1 Ser231 WT WT 25 EDL SOL mdKO 5 EDL SOL mdKO *** Main effect of genotype (p<0.01) Main effect of genotype (p<0.01) 20 ## 4 *
15 3 **
10 2 ***## Relative units Relative Relatvie units Relatvie
5 * 1 ### ### *** 0 0 Rest Contraction Rest Contraction Rest Contraction Rest Contraction
EDL# SOL# Rest# Contrac2on# Rest# Contrac2on# pTBC1D4 Ser711 H WT# mdKO# WT# mdKO# WT# mdKO# WT# mdKO# G pAMPK#Thr172# pAMPK#Thr172#
WT pACC#Ser212# pACC#Ser212# 3 EDL SOL mdKO pTBC1D1#Ser231# pTBC1D1#Ser231# ###* pTBC1D4#Ser711# pTBC1D4#Ser711# ##* pErk1/2#Thr202/Tyr204# pErk1/2#Thr202/Tyr204# 2 ACC# ACC#
TBC1D1# TBC1D1#
TBC1D4# TBC1D4#
Relative units Relative 1 Erk1/2# Erk1/2#
AMPKα1# AMPKα1#
** AMPKα2# AMPKα2#
0 Beta?ac2n# GAPDH# Rest Contraction Rest Contraction Page 31 of 37 FigureDiabetes 2
Delta Submaximal Insulin A EDL B Glucose uptake in EDL 50 §§§ Rest 20 # Rest §§§ §§§ Prior contraction Prior contraction
40 ### 15 *** 30 *** *** 10 20
5 [µmol / g protein h]
10 [µmol / g protein / h] 2-deoxyglucose uptake 2-deoxyglucose uptake 2-deoxyglucose
0 0 0 100 10.000 0 100 10.000 Insulin (µU/ml) WT mdKO
WT mdKO Delta Submaximal Insulin C SOL D Glucose uptake in SOL 50 Rest 35 Rest *** *** Prior contraction Prior contraction 30 40 25 30 20
20 15 10 [µmol / g protein / h] [µmol / g protein / h] 10 2-deoxyglucose uptake 2-deoxyglucose 2-deoxyglucose uptake uptake 2-deoxyglucose 5 0 0 0 100 0 100 Insulin (µU/ml) WT mdKO
WT mdKO
E WT F mdKO 110 * 110 p=0.1 * 0.3U/kg 0.3U/kg ** 100 100 *** Rest Rest Prior Exercise 90 Prior Exercise 90 20 25 80 80 15 20
70 ## 70 15 10 60 60 10 5 50 5
50 uptake 2-deoxyglucose 2-deoxyglucose uptake 2-deoxyglucose ng glucose / mg tissue / min / tissue mg / glucose ng 0 min / tissue mg / glucose ng 0 Bloodglucose Basal][% 10 Bloodglucose Basal][% 10 TA EDL 0 0 TA EDL 0 20 40 60 80 100 120 0 20 40 60 80 100 120 Time after insulin injection [min] Time after insulin injection [min] Rest 0.3 U/kg Prior exercise 0.3 U/kg Rest 0.3 U/kg Prior exercise 0.3 U/kg Rest 0.4 U/kg Prior exercise 0.4 U/kg Rest 0.4 U/kg Prior exercise 0.4 U/kg
G Area over the curve 0-40 min 600 Rest Prior contraction 500 # 400
300
200
Relative to Rest [%] Rest to Relative 100
0 WT mdKO FigureDiabetes 3 Page 32 of 37
A EDL B WT"SOL" WT mdKO Basal" Insulin" Prior contraction - + - + - + - + Prior"contracBon" !" +" !" +" Submaximal insulin - - + + - - + + Akt2" Akt2 TBC1D1" HKII
GS TBC1D4"
ACC ACC" PDH AMPKα2" TBC1D1 GAPDH" TBC1D4 GLUT4
AMPKα1 AMPKα2
GAPDH Page 33 of 37 FigureDiabetes 4
A EDL pAkt Thr308 B EDL pAkt Ser473 Rest 10 16 Prior contraction Rest *** *** 14 *** Prior contraction 8 12 *** 6 10 8 4 6 Relative units Relative Relative units Relative 4 2 2 0 0 0 100 0 100 Insulin (µU/ml) 0 100 0 100 Insulin (µU/ml)
WT mdKO WT mdKO C WT SOL pAkt Thr308 D WT SOL pAkt Ser473 8 Rest 8 Rest Prior contraction Prior contraction *** 6 6 ***
4 4 Relative units Relative 2 units Relative 2
0 0 0 100 0 100 Insulin (µU/ml) Insulin (µU/ml) FigureDiabetes 5 Page 34 of 37
A EDL pTBC1D4 Thr649 B EDL pTBC1D4 Ser711 4 Rest 3 Prior contraction ## Rest ### *** *** Prior contraction 3 *** 2 *** 2 *** ***
1 Relative units Relative 1 units Relative
0 0 0 100 0 100 Insulin (µU/ml) 0 100 0 100 Insulin (µU/ml) WT mdKO WT mdKO
C EDL pTBC1D4 Ser324 D EDL pTBC1D4 Ser595 Rest Rest 3 Prior contraction 5 Prior contraction *** *** 4 *** *** 2 3
2 1 Relative units Relative Relative units Relative 1
0 0 0 100 0 100 Insulin (µU/ml) 0 100 0 100 Insulin (µU/ml) WT mdKO WT mdKO
E Rest Prior contraction F Rest Prior contraction G Rest Prior contraction 30 30 3
20 20 2 r=0.43 r=0.47 r=0.65 p<0.05 p<0.05 p<0.001
10 10 1 Δ Glucose uptake Δ Glucose uptake [µmol / g protein h] [µmol / g protein h] Δ pTBC1D4 Thr649 [AU] 0 0 1 2 3 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 -0.5 0.0 0.5 1.0 1.5 2.0 Δ pTBC1D4 Thr649 [AU] Δ pTBC1D4 Ser711 [AU] Δ pTBC1D4 Ser711 [AU]
WT SOL pTBC1D4 Thr649 WT SOL pTBC1D4 Ser711 H Rest I Rest 7 Prior contraction 3 Prior contraction *** 6 *** 5 2 4 3 1 Relative units Relative 2 units Relative 1 0 0 0 100 0 100 Insulin (µU/ml) Insulin (µU/ml) Page 35 of 37 FigureDiabetes 6
A EDL pAMPK Thr172 B EDL pACC Ser212 1.5 1.5
Rest Rest Prior contraction Prior contraction 1.0 1.0
0.5 0.5 Relative units Relative Relative units Relative ***
0.0 0.0 0 100 0 100 Insulin (µU/ml) 0 100 0 100 Insulin (µU/ml)
WT mdKO WT mdKO
AMPKγ3-associated activity in EDL AMPKα2βγ1-associated activity in EDL C muscle 3 h after in situ contraction D muscle 3 h after in situ contraction 1.0 ## Rest 8 Rest Prior contraction Prior contraction ] ] p=0.13 -1 -1 0.8 6 mg mg ⋅ ⋅
-1 -1 0.6 4 min min ⋅ ⋅ 0.4 2 [ pmol
[ pmol 0.2
0.0 0 WT mdKO WT mdKO
WT Soleus WT Soleus AMPKα1-associated activity in EDL pAMPK Thr172 pACC Ser212 E muscle 3 h after in situ contraction F G 1.2 Rest 2.0 Rest 2.0 Rest Prior contraction Prior contraction Prior contraction ] 1.0 p=0.10 -1 1.5 1.5 mg
⋅ 0.8
-1 0.6 1.0 1.0 min ⋅ 0.4 Relative units Relative Relative units Relative 0.5 0.5
[ pmol 0.2
0.0 0.0 0.0 WT mdKO 0 100 0 100 Insulin (µU/ml) Insulin (µU/ml)
AMPK activity in WT SOL muscle H 2 h after in situ contraction I EDL pTBC1D4 Ser711 5 Rest Rest 2.0 p=0.07 Contraction ] Prior contraction # -1 4 1.5 p=0.28 mg ⋅
AMPK"WT" AMPKγ3"KO"
-1 3 !" +" !" +" Contrac=on" 1.0 pTBC1D4"Ser711" min ⋅ 2 TBC1D4" Relative units Relative 0.5 1 pACC"Ser212" [ pmol p=0.42 0.0 ACC" 0 AMPK WT AMPKγ3 KO AMPKγ3- AMPKα2βγ1- AMPKα1- associated associated associated FigureDiabetes 7 Page 36 of 37
A EDL pTBC1D1 Thr590 B EDL pTBC1D1 Ser231 2.5 Rest 2.0 Rest Prior contraction Prior contraction 2.0 *** *** 1.5 1.5 1.0 1.0 Relative units Relative Relative units Relative 0.5 0.5
0.0 0.0 0 100 0 100 Insulin (µU/ml) 0 100 0 100 Insulin (µU/ml)
WT mdKO WT mdKO
WT Soleus pTBC1D1 Thr590 WT Soleus pTBC1D1 Ser231 C Rest D Rest Prior contraction Prior contraction 2.5 2.0
2.0 *** 1.5 1.5 1.0 1.0 Relative units Relative Relative units Relative 0.5 0.5
0.0 0.0 0 100 0 100 Insulin (µU/ml) Insulin (µU/ml) Page 37 of 37 FigureDiabetes 8
A GS I-form B GS total activity
50 Rest 20 Rest Prior contraction *** Prior contraction 40 Main effect of prior contraction within mdKO (p<0.01) 15
30 *** 10 20 GS activity 5 10 GS activity: I-form (%) [mmol / mg protein / min] 0 0 0 100 0 100 Insulin (µU/ml) 0 100 0 100 Insulin (µU/ml)
WT mdKO WT mdKO C pGS 3a+3b D pGS 2+2a 1.5 Rest 1.5 Rest *** *** Prior contraction Prior contraction
1.0 1.0
0.5 0.5 Relative units Relative Relative units Relative
0.0 0.0 0 100 0 100 Insulin (µU/ml) 0 100 0 100 Insulin (µU/ml)
WT mdKO WT mdKO
Glycogen content in EDL muscle Glycogen content in WT SOL muscle E 3 h after in situ contraction F 2 h after in situ contraction 200 WT 150 mdKO
150 ## §§§ 100 100 ###
50 50 Glycosyl units [pmol / µg protein] 0 [pmol units protein] / µg Glycosyl 0 Rest Prior contraction Rest Prior contraction