muscle contraction during exercise is a more potent physiological stimulus of skeletal muscle glucose uptake than even maximal insulin   and a highly effective prophylactic against the hyperglycemia associated with the development of type 2 diabetes mellitus . despite these drug - target - wise attractive features , the molecular signalling mechanisms by which muscle contraction
augments glucose transport are , compared to insulin , relatively understudied and remain poorly defined ( for review see ref . ) .
the signals underlying increased glucose transport with contraction have long been speculated to include a feedforward signalling component , activated directly by depolarization - induced sr ca release through the ryanodine receptors . in addition , feedback signals are activated secondarily in response to ca - activated contraction to further fine - tune the glucose transport response , including probably activation of the metabolic stress responsive kinase 5 amp - activated protein kinase ( ampk ) by atp - turnover and mechanical stress - activated signals . the belief that ca by itself is adequate to stimulate glucose transport is based to a major extent on seminal work by john holloszy and co - workers where 34  mm caffeine was shown to increase sr ca release and glucose transport in incubated rat muscles without changes in muscle tension , atp or creatine phosphate . in rats , caffeine - stimulation was sufficient to double glucose transport rate in slow - twitch oxidative soleus muscle and triple it in fast - twitch glycolytic epitrochlearis   without activating ampk .
this suggested that ca 
 per se could stimulate a substantial increase in muscle glucose transport .
however , our group and others subsequently found 34  mm caffeine increased ampk activation and nucleotide - turnover in muscles from mice and rats [ 911 ] , presumably due to the considerable energy - demand posed by sarco / endoplasmatic reticulum ca atpase ( serca)-dependent ca reuptake .
furthermore , caffeine - stimulated glucose transport was potently inhibited in muscles from muscle - specific dominant - negative kinase - dead 2 ampk mice , suggesting that the caffeine - response largely depends on atp turnover - mediated activation of ampk rather than on ca as such . to clarify the relative sufficiency and necessity of sr ca vs. feedback signals to contraction - stimulated glucose transport
, we presently combined contractile myosin blockers , ampk transgenic mice , ca atpase inhibitors and electrical stimulated contraction , in incubated mouse muscles , a classical model system allowing cell culture - like manipulations and full environmental control of fully differentiated , contraction - competent , striated muscle .
all antibodies and reagents used were commercially available . details on antibodies used and immunoblotting specifics are included in supplemental experimental procedures .
soleus and edl muscles from anaesthetized female c57bl/6 wildtype and muscle - specific kd ampk overexpressing   mice were incubated in continuously gassed ( 95% o2/5% co2 ) modified krebs  ringer 
after 1015  min rest , buffer containing inhibitors was added for 1  h , followed by stimulation with cpa , insulin , electrical stimulation , aicar or passive stretch as described in the figure legends and in the supplemental experimental procedures .
changes in ca concentration were measured as fluo-3 fluorescence in confluent 3  h serum - starved l6 myotubes .
immunoprecipitated ampk trimer activity was measured in  vitro using p - labelled atp incorporation into a substrate peptide .
the data were analysed using t - test or one - way , two - way or two - way repeated measures anova with tukey 's post hoc test . the significance level was set at p  <  0.05 .
ex  vivo incubated mouse slow - twitch oxidative soleus and fast - twitch glycolytic extensor digitorum longus ( edl ) muscles were stimulated with the serca - inhibitor cyclopiazonic acid ( cpa ) at increasing doses to inhibit ca reuptake , thus allowing a sustained ca leak from the sarco / endoplasmatic reticulum ( sr ) .
in soleus muscle , this caused a dose - dependent increase in the ca - regulated thr57 phosphorylation of eef2 by eef2 kinase , a readout of sr ca release , in addition to increased phosphorylation of ampk thr172 ( figure  1a ) .
this pattern is reminiscent of the increase in eef2 and ampk phosphorylation seen in soleus stimulated with 46  mm caffeine ( figure  s1a ) .
glucose transport was elevated significantly above baseline using 50  m cpa ( figure  1b ) .
no effect of cpa was observed in mouse edl ( figure  1a ) , probably due to the known differences in ca handling proteins between type ii fibres compared to type i fibres .
pretreatment with the sr ca channel blocker dantrolene prevented both the cpa - stimulated eef2 and ampk phosphorylations ( figure  1c ) and directly measured ca release in l6 myotubes ( figure  1d ) , showing their dependence on sr ca release .
the fast and slow contractile myosin heavy chain type ii atpase inhibitors , bts and blebbistatin ( bleb ) did not affect ca release ( figure  1d ) consistent with previous reports [ 1618 ] .
the cpa - induced rise in cytosolic ca leak elicited a sustained submaximal muscle contraction ( figure  1e ) .
inhibition of contractile function by bts  +  bleb blocked tension development ( figure  1e  +  dose  response curves in figure  s1b ) , caused a general increase in the creatine phosphate / creatine ( crp / cr)-ratio ( figure  1f ) and markedly reduced cpa - induced metabolic stress measured as lactate concentration ( figure  1 g ) .
we were initially concerned that bleb in particular might have off - target effects since myoii has been implicated in insulin - stimulated glut4 translocation and glucose transport in adipocytes .
however , neither insulin nor aicar - stimulated signalling ( figure  2a+c ) or glucose transport ( figure  2b+d ) were inhibited by bts  +  bleb , suggesting that the effects of myosin atpase inhibition are specific to contraction .
myosin atpase inhibition in soleus muscle potently dampened the cpa - induced phosphorylation of ampk and p38 mapk , ( figure  2a ) activated by atp - turnover   and mechanical stress , respectively .
bts  +  bleb significantly inhibited cpa - stimulated 2 ampk activity and had a qualitatively similar effect on 1 ampk activity ( figure  s1c ) .
myosin atpase blockade potently reduced cpa - stimulated glucose transport ( figure  3 ) .
next , we asked if the residual cpa - stimulated glucose transport - response in bts  +  bleb - treated soleus muscles required ampk signalling . as expected , our signalling readouts including the phosphorylation of ampk by upstream kinases on thr172 were not affected by muscle - specific overexpression of kinase - dead 2 ( kd ) ampk ( figure  3b ) .
cpa - stimulated glucose transport was unaffected by muscle - specific overexpression of kd ampk ( figure  3e ) .
similar to the findings with bts  +  bleb in figure  3a , combining bts  +  bleb with kd ampk inhibition only reduced ampk thr172 and p38 mapk thr180/tyr182 phosphorylation , but did not affect eef2 thr57 phosphorylation ( figure  3c ) . interestingly , despite the normal sr ca dependent signalling , the residual glucose transport - response in the presence of bts  +  bleb was completely abolished in the presence of kd ampk ( figure  3f ) .
no effect of cpa or bts  +  bleb was found for total protein expression ( figure  s1d ) .
overall , these data show that cpa - induced contraction - stimulated glucose transport can be completely prevented despite normal sr ca release .
rather , this appears to require input from ampk and likely mechanical stress - signalling .
a sustained increase in cytosolic ca by cpa is quite dissimilar from the transient ca spikes elicited by physiological muscle excitation  contraction coupling .
therefore , we also investigated the effect of contractile inhibition on the glucose transport - response elicited by intermittent electrical field stimulation of muscle contraction . given that two previous reports saw widely different effect of bts on the muscle glucose transport - response in rodent fast - twitch muscles ( 50% reduction vs. no effect ) , these previously published stimulation protocols were included in our experimental setup and directly compared to a third less intense protocol ( representative force - traces shown in figure  4a ) . with these protocols ,
muscles were stimulated for 0.1% , 0.3% and 0.7% of the total time ( % net stimulation time ( nst ) ) and a clear intensity - dependent effect on fatigue rates measured for the initial 3  min was observable ( figure  4b ) .
the tension development during electrical stimulation was all but prevented by the addition of myosin atpase inhibitors ( figure  4b ) .
inhibition of cross - bridge cycling markedly reduced metabolic stress measured as crp / cr - ratio ( figure  4c ) and muscle lactate ( figure  4d ) .
the effect of myosin atpase inhibition on the electrically stimulated glucose transport - response was highly dependent on the stimulation protocol , ranging from no difference in glucose transport - stimulation when applying the most intense 0.7% nst protocol in edl to a complete prevention of glucose transport using the mild 0.1% nst protocol in both soleus and edl muscles ( figure  5a+b ) .
consistent with our cpa - experiments , the electrically stimulated increases in eef2 thr57 phosphorylation were unaffected by myosin atpase inhibitors whereas the increases in ampk thr172 and p38 mapk thr180/tyr182 phosphorylation were markedly blunted by inhibition of contraction by the myosin inhibitors ( figure  5c ) .
no effect of ex  vivo contraction or bts  +  bleb was observed for total protein expression ( figure  s2a ) .
ampk kinase activity measurements on subsets of ampk trimer complexes in  vitro revealed that the residual increase in ampk thr172 phosphorylation with myosin atpase inhibitors reflected a normal increase of 1 ampk associated trimer activity but a very potent inhibition of the 223 ampk trimer ( figure  5d ) .
the decreased 223 ampk activation was associated with decreased phosphorylation of tbc1d1 ser231 and accbeta ser212 ( figure  5e ) , previously suggested to be 223 ampk substrates in exercising humans . using more intense 0.3% and 0.7% nst stimulation , no discernible difference in tbc1d1 ser231 phosphorylation
was observed between control and bts  +  bleb - treated muscles ( figure  s2b ) . since the residual cpa - stimulated glucose transport - response in the presence of bts  +  bleb is ampk - dependent ,
this was also tested for electrical stimulation . given that bts  +  bleb - treated muscles only increase glucose transport in response to intense stimulation , we asked if the residual glucose transport - response to the most intense 0.7% nst stimulation was reduced in kd ampk muscles .
however , no significant effect of kd ampk expression was seen on the residual increase in glucose transport stimulated with the 0.7% protocol ( figure  s2c ) . still , similar to cpa - stimulated glucose transport , the experiments using low intensity 0.1% nst electrical stimulation define conditions where contraction - stimulated glucose transport is abolished ( figure  5a+b ) and associated with decreased 223 ampk ( figure  5d ) and mechanical stress - signalling but not increased sr ca on its own  ( figure  5c ) .
stimulation of ampk activity and application of mechanical stress ( i.e. passive stretch ) have both been shown to stimulate glucose transport in incubated rodent muscles without affecting indices of sr ca release .
however , the interaction between these stimuli is not known , nor is it clear how much of the total contraction - stimulated glucose transport - response is rallied by combined stimulation with ampk and mechanical stress . to test this , a series of additivity - experiments
we first performed aicar and passive stretch additivity experiments in soleus and edl muscles from wildtype and kd ampk expressing mice .
ampk and p38 mapk phosphorylations were increased by aicar and passive stretch , respectively , whereas neither stimulus altered eef2 phosphorylation ( figure  6a+b ) .
ampk - dependent phosphorylation of acc2 was blocked in kd ampk expressing muscles ( figure  6a+b ) .
no effect of aicar or stretch were observed on total protein expression ( figure  s3 ) .
glucose transport was increased by passive stretch in both muscles ( figure  6c+d ) whereas aicar only increased glucose transport significantly in edl muscle ( figure  6d ) . in both soleus and edl , combined aicar  +  stretch additively stimulated glucose transport compared to aicar or stretch alone ( figure  6c+d ) .
overexpression of kd ampk abolished aicar but not stretch - stimulated effects on glucose transport ( figure  6c+d ) , confirming that stretch - stimulated glucose transport is ampk - independent .
overall , these experiments support a model where ampk activation and stretch independently and additively increase glucose transport . to directly compare the aicar  +  stretch - stimulated glucose transport - response to contraction and insulin - stimulated glucose transport , aicar  +  stretch - stimulation
was further combined with either insulin or intermediate intensity 0.3% nst electrically stimulated contraction in the absence or presence of the phosphatidylinositol-3-kinase ( pi3k ) inhibitor wortmannin . signalling - wise , akt ser474
was only increased by insulin and eef2 thr57 phosphorylation was only increased by contraction ( figure  7a+b ) .
ampk and p38 mapk phosphorylation responses were generally as expected , apart from some unexpected interactions with wortmannin , including potentiation of contraction - stimulated ampk activation by wortmannin and a strong inhibitory effect of wortmannin on p38 mapk phosphorylation by stretch ( figure  7a+b ) .
similar to the contraction - response , the glucose transport - response to aicar  +  stretch was insensitive to wortmannin in both soleus ( figure  7c left ) and edl ( figure  7c right ) muscles .
furthermore , aicar  +  stretch - stimulated glucose transport was fully additive to insulin- but not contraction - stimulated glucose transport .
lastly , the glucose transport - response to aicar  +  stretch was comparable in magnitude to the contraction - stimulated glucose transport - response in both muscles ( figure  7c ) .
these results show that aicar  +  stretch can fully mobilize the full contraction - stimulated glucose transport - response without increasing sr ca dependent signalling .
similar to contraction , this response is additive to insulin - stimulation and pi3k - independent .
stretch - stimulated glucose transport has previously been suggested to be mediated by p38 mapk .
complete blockade of p38 mapk - dependent signalling to hsp27 using the novel p38 mapk inhibitor indicated the effectiveness of the blocker vx-702 but it did not reduce stretch - stimulated glucose transport ( figure  s4 ) , indicating that p38 mapk may not be essential to stretch - stimulated glucose transport .
the premise that sr ca release per se increases glucose transport in muscle has remained largely undisputed and continues to be reiterated and built upon in the scientific literature .
however , here we demonstrated that 1 ) sr ca release is unable to increase glucose transport in the absence of muscle contraction - related feedback signals and 2 ) that the full contraction - response in glucose transport can be obtained without increased sr ca dependent signalling .
apart from disputing what has become a text - book paradigm , this study is to our knowledge the first study to completely block and thus account for 100% of the glucose transport - response during contraction induced by either cpa or electrical stimulation and to reconstitute 100% of the contraction - response by combining two of its proposed components , ampk activation and mechanical stress .
muscle contraction is a complex stimulus that activates a myriad of independent signalling pathways that may regulate a given endpoint . over the years
, this has made it extremely challenging to convincingly assign a given signalling protein to glucose transport - regulation by contraction .
our current work shows that the parallel and independent inputs from ampk and mechanical stress are likely both required for glucose transport - stimulation and sufficient to recruit the full contraction - sensitive glucose transport response .
any protein partly required for contraction - stimulated glucose transport independently of ampk is a candidate for regulating stretch - stimulated glucose transport , including rac1 , reactive oxygen species   and the ampk - related kinase snark .
a number of studies in isolated rodent muscles have proposed that various ca - regulated proteins are necessary for the full contraction - stimulated glucose transport - response , including pkc isoforms [ 2830 ] , ca / calmodulin - dependent kinase ( camk)ii , nspl1   and camkk . from our study ,
sr - ca release is clearly neither sufficient nor required to elicit the full contraction - like glucose transport - response .
worth noting , however , aicar stimulation takes 40  min to maximally stimulate glucose transport while ex  vivo contraction - stimulated glucose transport peaks within 510  min .
therefore , other signals , including perhaps ca , may accelerate the completion of the glucose transport - response during contraction .
furthermore , our data do not rule out a contribution from other ca sources , most notably extracellular ca entry through voltage and/or stretch - sensitive plasma membrane ca channels . silencing of the transient receptor potential ( trp ) 3 ca channel in mouse flexor digitorum brevis single muscle fibres reduced insulin - stimulated glucose transport , suggesting a role of extracellular ca in insulin - stimulated glut4 vesicle membrane fusion .
interestingly , pharmacological or stretch - stimulated trp3-mediated ca entry resulted in increased activation of camkii , rac1 and ros production in mouse heart , all of which have previously been linked to contraction - stimulated glucose transport . however , pharmacological activator and inhibitor - studies do not support an essential role of trp channels during skeletal muscle contraction . for the voltage - gated l - type ca channels , inhibitor - specificity may limit conclusions regarding their requirement for muscle glucose transport . based on these studies , a contribution from other surface ca channels and non sr ca dependent activation of for instance
bts was first characterized in 2001 as a specific inhibitor of the fast - twitch ( myosin heavy chain ( mhc ) type ii ) , but not slow - twitch muscle / cardiac myoii ( mhc type i ) isoform .
remarkably , concentrations as high as 100  m bts did not affect the activity of any of the enzymes tested including kinesin , pyruvate kinase , lactate dehydrogenase , non - muscle human platelet myoiia nor did it affect electrically stimulated ca transients .
a few years later , bleb was found to specifically inhibit non - muscle myoiia and b , in addition to skeletal muscle fast - twitch and porcine slow - twitch heart myoii without effects on other myosin classes .
also , bleb inhibited the contractility of mouse cardiomyocytes without affecting action potentials or ca entry . still , we feared that bleb might have off - target effects since myoii has been implicated in insulin - stimulated glut4 translocation and glucose uptake in adipocytes [ 19,4244 ] .
however , no effect of bts  +  bleb treatment was observed on insulin or aicar - stimulated signalling or glucose transport .
moreover , the decline in glucose transport - response with bts  +  bleb was absent in edl at the highest intensity 0.7% nst contraction - intensity , despite a sustained effect on tension development .
therefore , an acute effect of myoii inhibition on glucose transport or on glut4 independently of cross - bridge cycling is unlikely .
our data also indicate that myoii isoforms are dispensable to both insulin and aicar - stimulated glucose transport in mouse skeletal muscle .
to compare different muscle types , our study encompassed both the relatively slow - twitch oxidative soleus and fast - twitch glycolytic edl muscle .
with contraction , both muscles displayed qualitatively similar increments in glucose transport with increasing contraction - intensity , demonstrating that the dependence on tension development to stimulate glucose transport holds true for both slow twitch and fast twitch fibre types .
this agrees with earlier conclusions in incubated rat muscles , where glucose transport correlated with force - production and metabolic stress rather than contraction - frequency .
the response to cpa differed markedly between the two muscle types , with a lack of cpa - stimulated eef2 phosphorylation in edl .
a more sensitive tension - development in slow vs. fast - twitch muscle in response to ca has been described in rodents previously . from a molecular viewpoint
, this difference may relate to the relatively more extensive sr in fast - twitch fibres , the exclusive expression of the cytosolic ca buffer protein , parvalbumin and a less ca sensitive troponin c - isoform , in addition to higher expression of ca handling proteins such as dhpr1s , ryr1 and serca1 ( for references see e.g. ) . at higher rates of ca release , specifically when
combining cpa with caffeine , our group has previously reported increased tension - development , eef2 and ampk phosphorylation in rat edl .
however , we have repeatedly observed that glucose transport is reduced , not increased , by cpa  +  caffeine - treatment in mouse edl ( unpublished observation ) , perhaps associated with triggering of cell necrosis or apoptosis by elevated ca .
therefore , the cpa - studies were limited to soleus muscle . nonetheless , our electrical stimulation - data allowed us to demonstrate a similar uncoupling of sr ca activated signalling and glucose transport in both soleus and edl muscles .
overall , these data indicate that both slow - twitch oxidative and fast - twitch glycolytic muscles depend mainly on signals secondary to cross - bridge cycling to elicit contraction - stimulated glucose transport .
unexpectedly , the inhibitory effect of bts  +  bleb on contraction - stimulated glucose transport varied from complete inhibition at the lowest intensity 0.1% nst electrical stimulation protocol to no effect in edl during stimulation with the highest intensity 0.7% nst protocol .
firstly , different electrical stimulation protocols provide different answers , implying that ideally several different electrical stimulation protocols should be evaluated for a given intervention .
secondly , the less potent bts  +  bleb inhibition at higher intensities could indicate either that glucose transport at higher intensities relies on distinct mechanisms not related to tension - development as proposed previously , or that electrical stimulation - related artefacts such as ros - production by electrolysis   increasingly mobilize compensatory mechanisms . whether such compensatory mechanisms during intense electrical field stimulation are physiologically meaningful or rather electrical stimulation artefacts is presently unknown . based on studies using pharmacological activators , ampk is necessary ( see   for references ) and likely sufficient   to stimulate glucose transport in skeletal muscle , in particular those dominated by type ii fibres .
thus , ampk activation can clearly increase glucose transport , with the downstream mechanisms proposed to involve tbc1d1 , no synthases , pikfyve , nspl1   and/or the rala gap protein garnl1 , with rala previously being linked to glut4 translocation in muscle downstream of rac1 .
however , the contribution of ampk during contraction - stimulated glucose transport remains somewhat controversial .
thus , investigations by various groups in the same model as presently used , the kinase - dead 2 ampk overexpressing mouse , have found partly decreased , normal or increased contraction or exercise - stimulated glucose uptake in this model compared to wildtype ( see   for references ) . adding to the uncertainty , double knockout of both regulatory ampk -subunits abolished ampk - activity and potently reduced contraction- and treadmill running stimulated glucose transport   whereas double knockout of the catalytic ampk -subunits did not affect contraction - stimulated glucose transport . in the present study ,
passive stretch and aicar additively increased glucose transport , and the residual 1/3rd of cpa - stimulated glucose transport that was not blocked by bts  +  bleb , was abolished in the kd ampk mice .
thus , while ampk activation can clearly blend with other signals to elicit the full contraction - stimulated glucose transport - response , the contribution from ampk during contraction may be hard to isolate in various ko models due to compensation and redundancy of other glucose transport - stimulating signals during contraction . in conclusion ,
combined ampk activation and mechanical stress can fully account for the contraction - stimulated glucose transport - response ex  vivo , strongly suggesting that ampk and stretch - activated signalling , but not sr ca 
 per se , regulates glucose transport during skeletal muscle contraction .
 understanding how muscle contraction orchestrates insulin - independent muscle glucose transport may enable development of hyperglycemia - treating drugs . 
 the prevailing concept implicates ca2 + as a key feed forward regulator of glucose transport with secondary fine - tuning by metabolic feedback signals through proteins such as ampk . here , we demonstrate in incubated mouse muscle that ca2 + release is neither sufficient nor strictly necessary to increase glucose transport . 
 rather , the glucose transport response is associated with metabolic feedback signals through ampk , and mechanical stress - activated signals . 
 furthermore , artificial stimulation of ampk combined with passive stretch of muscle is additive and sufficient to elicit the full contraction glucose transport response . 
 these results suggest that atp - turnover and mechanical stress feedback are sufficient to fully increase glucose transport during muscle contraction , and call for a major reconsideration of the established ca2 + centric paradigm . 
Introduction
Materials and methods
Results
Discussion