protocols complied with the guidelines of the institutional animal care and use committee of the joslin diabetes center and the national institutes of health . male imprinting control region ( icr ) mice ( aged 710 weeks ) ( taconic , hudson , ny ) , ampk2 inactive transgenic mice on an fvb background , and matched controls were studied ( 27 ) . the construction of glut4-egfp has been described previously ( 16,29 ) .
the skin was opened to expose the quadriceps muscle to the bombardment of dna / gold particles using the helios gene gun system .
each muscle was shot twice with 200 psi pressure with 1 g dna/0.5 mg gold ( 0.6 m gold particle size ) .
after transfection , the skin was closed using sutures and the animals were allowed to recover .
five days after transfection , mice were anesthetized and mounted in dental cement ( 5,29 ) . for contraction experiments , immediately prior to mounting the mice , microelectrodes were placed into the superficial muscle fiber layer near the groin and knee region of the quadriceps muscle , producing single twitch contractions only in the superficial layers of the quadriceps muscle fibers without losing imaging focus .
such stimulation activates muscle fibers via stimulation of the motor nerve fibers and motor endplates .
stimulation was not applied directly to the nerve because it resulted in either no visually detectable contractions or entire muscle belly contractions resulting in loss of optical focus during contraction . for aicar stimulation ,
the mouse was mounted , a catheter was placed in a tail vein , and a single confocal image was taken .
this was followed by an additional 30-min stabilization period followed by another basal image ( t = 0 ) .
for the contraction protocol , immediately after the second basal image ( t = 0 ) , direct electrical stimulation to the muscle fibers was initiated .
two contraction treatments of different intensities were used : 1 ) contractions elicited by lower voltage and the parameters were frequency 2 hz , duration 60 milliseconds , voltage 0.10.9 v , consisting of three bouts of 10 min , separated by 90 s of rest and 2 ) contractions elicited by higher voltage and the parameters were frequency 2 hz , duration 60 milliseconds , voltage 1.13.0 v , consisting of three bouts of 5 min , separated by 90 s of rest . both protocols elicited repeated single twitch contractions .
protocols were selected based on empirical observations of contraction force taken together with what was possible without losing optical focus of the imaged muscle fiber .
we did not use an increasing contraction frequency because it made it very difficult to continually monitor fiber contraction . in our experience ,
the voltage field intensity generated by a higher voltage induced visually greater fiber contraction amplitude .
this difference in contraction intensity is what we refer to as high- or low - voltage contractions throughout the study .
contractions of the imaged fibers were continually monitored during the contraction periods through the oculars of the microscope , and voltage was adjusted to maintain force .
images were collected at the end of each contraction period and every 10-min post - contraction up to 130 min .
the imaging of a high - voltage in situ contraction period is shown in supplementary video 1 ( available online at http://diabetes.diabetesjournals.org/cgi/content/full/dc10-0233/dc1 ) , illustrating that clear images are not obtainable when the fibers are contracting .
for aicar stimulation , aicar was given as an intravenous bolus injection ( 1 g / kg ) through the tail vein , and images were collected every 10 min for 240 min .
images were collected with a 63 , 1.2 na zeiss c - apochromat objective on a zeiss - lsm-410 confocal microscope with arkr-488 nanometer ( nm ) laser line for excitation of egfp , while green emission light was collected between 500 and 530 nm using the standard fluorescein isothiocyanate filter .
sulforhodamine b images were collected as described ( 5 ) . the tagged image file format ( tiff )
images obtained with the zeiss confocal software were imported into metamorf software ( version 6.1 ; universal imaging ) .
since it has previously been reported that glut4 translocation and re - internalization in muscle are products of endocytosis and exocytosis movements , we measured the net amount of glut4-egfp at the various time points ( 31 ) .
the net amount of glut4-egfp at membrane surfaces was quantified using metamorf software by drawing thin regions of interest ( rois ) at the sarcolemma and t - tubule regions , excluding vesicle structures , as previously described ( 5,15,16 ) .
briefly , intracellular vesicle depots were quantified by measuring changes in gray value intensity in glut4-egfp vesicles larger than 2 m in the visible fiber area inside an roi covering both sarcolemma and t - tubules ( fig .
the threshold and classifying settings in the metamorf software were used to discriminate depots above 2 m in size from the background and emerging staining of sarcolemma and t - tubules .
these metamorf settings were also used to further discriminate between perinuclear and nonperinuclear glut4-egfp depots .
for sarcolemma , measurements from both sarcolemma edges of the confocal picture were averaged , the perinuclear areas were excluded , and measurements were normalized to the area of the sarcolemma roi ( fig .
for t - tubules , measurements from four randomly chosen areas were averaged ( fig .
1b ) . to compensate for variation in glut4-egfp expression among fibers , the arbitrary units shown are the actual gray value of roi fluorescence divided by roi fluorescence value at t = 0 ( f / f0 ) .
contraction - mediated glut4-egfp translocation to both sarcolemma and t - tubules . a and b : examples of the regions of interest ( rois ) used for image quantifications .
a : fluorescence values of glut4-egfp perinuclear and nonperinuclear depots were measured within the same roi .
quantification software settings were set to measure vesicles > 2 m in size inside the roi and to discriminate between perinuclear and nonperinuclear depots .
b : the level of glut4-egfp translocation in the muscle fibers at the different time points was measured in rois along each sarcolemma side and in four randomly chosen rois in the t - tubules region .
c : in vivo staining of t - tubules after intravenous injection of the water soluble dye sulforhodamine b , showing a high fluorescence staining in the capillaries running along the sarcolemma together with a more weakly fluorescent striated pattern in the t - tubules region .
d and e : t = 0 shows confocal images of basal glut4-egfp  expressing icr mouse muscle fibers just before in situ contractions .
muscle was stimulated for 3  5 min ( d ) or 3  10 min ( e ) periods separated by 90 sec of rest .
images are shown from one muscle fiber subjected to either high- ( d ) or low - voltage ( e ) contractions .
horizontal arrows indicate sarcolemma , while glut4-egfp depots are indicated near the nuclei by vertical arrows and inside the fiber by diagonal arrows .
f - h : image quantifications of glut4-egfp at the sarcolemma ( f ) , the t - tubules ( g ) , and the intracellular vesicle depots ( h ) .
* statistical difference between groups ( p < 0.05 ) in values obtained at the end of identical contraction bouts .
arbitrary units shown are the actual grey value of roi fluorescence divided by roi fluorescence value at t = 0 ( f / f0 ) .
n = 58 . to verify the striated appearance and orientation of the t - tubules , we used sulforhodamine b , a water soluble dye that is not taken up by muscle fibers but will fill and stain the lumen of the t - tubules ( 3234 ) ( fig .
sulforhodamine b was injected into the tail vein , and a confocal picture of quadriceps fibers was obtained 3 min later when the dye had fully loaded the t - tubules .
staining resulted in two very strong vertical florescent lines representing the capillaries and interstitial space near the sarcolemma and fine striated lines indicating the lumen of the t - tubules .
the difference in the intensity of the sulforhodamine b staining is due to the much smaller diameter of the t - tubule lumen ( 100 nm ) compared with the flanking capillaries ( 40 m ) and interstitial space .
this striated pattern for t - tubules is consistent with previous findings ( 4,5,15,35 ) .
mice were briefly anesthetized with 90 mg / kg pentobarbital i.p . , and the skin was opened to expose the quadriceps muscle to the bombardment of dna / gold particles using the helios gene gun system .
each muscle was shot twice with 200 psi pressure with 1 g dna/0.5 mg gold ( 0.6 m gold particle size ) .
after transfection , the skin was closed using sutures and the animals were allowed to recover .
five days after transfection , mice were anesthetized and mounted in dental cement ( 5,29 ) . for contraction experiments , immediately prior to mounting the mice , microelectrodes were placed into the superficial muscle fiber layer near the groin and knee region of the quadriceps muscle , producing single twitch contractions only in the superficial layers of the quadriceps muscle fibers without losing imaging focus .
such stimulation activates muscle fibers via stimulation of the motor nerve fibers and motor endplates .
stimulation was not applied directly to the nerve because it resulted in either no visually detectable contractions or entire muscle belly contractions resulting in loss of optical focus during contraction . for aicar stimulation ,
the mouse was mounted , a catheter was placed in a tail vein , and a single confocal image was taken .
this was followed by an additional 30-min stabilization period followed by another basal image ( t = 0 ) .
for the contraction protocol , immediately after the second basal image ( t = 0 ) , direct electrical stimulation to the muscle fibers was initiated .
two contraction treatments of different intensities were used : 1 ) contractions elicited by lower voltage and the parameters were frequency 2 hz , duration 60 milliseconds , voltage 0.10.9 v , consisting of three bouts of 10 min , separated by 90 s of rest and 2 ) contractions elicited by higher voltage and the parameters were frequency 2 hz , duration 60 milliseconds , voltage 1.13.0 v , consisting of three bouts of 5 min , separated by 90 s of rest . both protocols elicited repeated single twitch contractions .
protocols were selected based on empirical observations of contraction force taken together with what was possible without losing optical focus of the imaged muscle fiber .
we did not use an increasing contraction frequency because it made it very difficult to continually monitor fiber contraction . in our experience , the voltage field intensity generated by a higher voltage induced visually greater fiber contraction amplitude .
this difference in contraction intensity is what we refer to as high- or low - voltage contractions throughout the study .
contractions of the imaged fibers were continually monitored during the contraction periods through the oculars of the microscope , and voltage was adjusted to maintain force .
images were collected at the end of each contraction period and every 10-min post - contraction up to 130 min .
the imaging of a high - voltage in situ contraction period is shown in supplementary video 1 ( available online at http://diabetes.diabetesjournals.org/cgi/content/full/dc10-0233/dc1 ) , illustrating that clear images are not obtainable when the fibers are contracting .
for aicar stimulation , aicar was given as an intravenous bolus injection ( 1 g / kg ) through the tail vein , and images were collected every 10 min for 240 min .
images were collected with a 63 , 1.2 na zeiss c - apochromat objective on a zeiss - lsm-410 confocal microscope with arkr-488 nanometer ( nm ) laser line for excitation of egfp , while green emission light was collected between 500 and 530 nm using the standard fluorescein isothiocyanate filter .
the tagged image file format ( tiff ) images obtained with the zeiss confocal software were imported into metamorf software ( version 6.1 ; universal imaging ) .
since it has previously been reported that glut4 translocation and re - internalization in muscle are products of endocytosis and exocytosis movements , we measured the net amount of glut4-egfp at the various time points ( 31 ) .
the net amount of glut4-egfp at membrane surfaces was quantified using metamorf software by drawing thin regions of interest ( rois ) at the sarcolemma and t - tubule regions , excluding vesicle structures , as previously described ( 5,15,16 ) .
briefly , intracellular vesicle depots were quantified by measuring changes in gray value intensity in glut4-egfp vesicles larger than 2 m in the visible fiber area inside an roi covering both sarcolemma and t - tubules ( fig .
the threshold and classifying settings in the metamorf software were used to discriminate depots above 2 m in size from the background and emerging staining of sarcolemma and t - tubules .
these metamorf settings were also used to further discriminate between perinuclear and nonperinuclear glut4-egfp depots . for sarcolemma ,
measurements from both sarcolemma edges of the confocal picture were averaged , the perinuclear areas were excluded , and measurements were normalized to the area of the sarcolemma roi ( fig .
1b ) . to compensate for variation in glut4-egfp expression among fibers , the arbitrary units shown are the actual gray value of roi fluorescence divided by roi fluorescence value at t = 0 ( f / f0 ) .
a and b : examples of the regions of interest ( rois ) used for image quantifications .
a : fluorescence values of glut4-egfp perinuclear and nonperinuclear depots were measured within the same roi .
quantification software settings were set to measure vesicles > 2 m in size inside the roi and to discriminate between perinuclear and nonperinuclear depots .
b : the level of glut4-egfp translocation in the muscle fibers at the different time points was measured in rois along each sarcolemma side and in four randomly chosen rois in the t - tubules region .
c : in vivo staining of t - tubules after intravenous injection of the water soluble dye sulforhodamine b , showing a high fluorescence staining in the capillaries running along the sarcolemma together with a more weakly fluorescent striated pattern in the t - tubules region .
d and e : t = 0 shows confocal images of basal glut4-egfp  expressing icr mouse muscle fibers just before in situ contractions .
( 1.13 v ) ( d ) or  low  ( 0.10.9 v ) ( e ) voltage . muscle was stimulated for 3  5 min ( d ) or 3  10 min ( e ) periods separated by 90 sec of rest .
images are shown from one muscle fiber subjected to either high- ( d ) or low - voltage ( e ) contractions .
horizontal arrows indicate sarcolemma , while glut4-egfp depots are indicated near the nuclei by vertical arrows and inside the fiber by diagonal arrows .
f - h : image quantifications of glut4-egfp at the sarcolemma ( f ) , the t - tubules ( g ) , and the intracellular vesicle depots ( h ) .
* statistical difference between groups ( p < 0.05 ) in values obtained at the end of identical contraction bouts .
arbitrary units shown are the actual grey value of roi fluorescence divided by roi fluorescence value at t = 0 ( f / f0 ) .
n = 58 . to verify the striated appearance and orientation of the t - tubules , we used sulforhodamine b , a water soluble dye that is not taken up by muscle fibers but will fill and stain the lumen of the t - tubules ( 3234 ) ( fig .
sulforhodamine b was injected into the tail vein , and a confocal picture of quadriceps fibers was obtained 3 min later when the dye had fully loaded the t - tubules .
staining resulted in two very strong vertical florescent lines representing the capillaries and interstitial space near the sarcolemma and fine striated lines indicating the lumen of the t - tubules .
the difference in the intensity of the sulforhodamine b staining is due to the much smaller diameter of the t - tubule lumen ( 100 nm ) compared with the flanking capillaries ( 40 m ) and interstitial space .
this striated pattern for t - tubules is consistent with previous findings ( 4,5,15,35 ) .
values obtained at the end of the various contraction bouts ( fig . 1 ) and calculated areas under the curve ( fig .
in the basal state , glut4-egfp was found in larger and smaller vesicle structures ( fig .
1d and e ) ( t = 0 , diagonal arrow ) and in the perinuclear region ( t = 0 , vertical arrow ) , as has been previously reported ( 4,5,16,29,36 ) .
1d and e ) ( t = 0 , horizontal arrow ) and no regular striated pattern throughout the fiber . following basal imaging ,
the superficial fibers of the quadriceps were contracted with high or low voltage . in mice subjected to high voltage contractions ,
there was marked staining along the sarcolemma by the 5 min time point ( fig .
1d ) ( t = 5 ) , quantified as a 60% increase above basal ( fig .
a striated pattern emerged inside the muscle fibers by 5 min showing translocation to the t - tubules ( fig .
, there was a 90% increase in t - tubule glut4-egfp after 5 min of contraction ( fig .
, there was a marked depletion of glut4-egfp vesicles in intracellular glut4-egfp vesicle depots , which also included the perinuclear region ( fig .
two additional 5-min contraction periods further increased glut4-egfp staining of sarcolemma and t - tubules ( > 200% increase from basal ) simultaneously with a further reduction in glut4-egfp vesicle depots ( 70% decrease from basal ) ( fig .
the depot depletion corresponded to a 75% maximal reduction in nonperinuclear depots and a 65% maximal reduction in perinuclear depots . with contractions elicited by low voltage
, there was a significant but modest glut4-egfp translocation to the sarcolemma by 10 min , which appeared in the form of continuous glut4-egfp staining ( fig .
the increases in glut4-egfp at the sarcolemma and t - tubules were accompanied by a 30% decrease in intracellular glut4 vesicle depots ( fig .
translocation to the sarcolemma and t - tubules steadily increased , and glut4-egfp vesicle depots were further reduced ( fig .
1e [ t = 20 , t = 30 , diagonal arrow ] and f  h ) .
1e ) ( t = 30 , horizontal arrow ) and t - tubules ( fig .
1e ) ( t = 30 , striated pattern ) occurred at the same time , indicating that glut4 translocation to these distinct cell surface membranes followed similar kinetics ( fig . 1f 
this is in contrast to what we have previously reported for insulin - mediated glut4-egfp translocation ( 5 ) .
comparison of high- and low - voltage contraction revealed that the high - voltage protocol resulted in greater levels of glut4-egfp translocation to the sarcolemma and t - tubules and a greater decrease in glut4 vesicle depots ( fig .
we next investigated the kinetics of glut4-egfp re - internalization in the period following muscle contractions , using the 3  5 min high - voltage contraction protocol that resulted in maximal translocation .
1 ) , glut4-egfp translocation to the sarcolemma and t - tubules increased gradually throughout the contractions , and there was a concomitant decrease in intracellular depots ( fig .
2 ) . interestingly , peak glut4-egfp depot depletion had occurred by 10 min ( fig .
2a [ t = 10 ] and d ) , whereas glut4-egfp translocation to the sarcolemma and t - tubules continued to increase during the first 10 min following the cessation of contractions ( fig .
this indicates a delay in the arrival of glut4-egfp at the sarcolemma and t - tubules . following peak translocation to the sarcolemma and t - tubules , glut4-egfp underwent gradual net re - internalization and was almost fully re - internalized by 130 min after contractions ( fig .
the time course of glut4-egfp re - internalization was similar for the sarcolemma and t - tubules ( fig .
c ) . in situ contractions induce glut4-egfp translocation to and re - internalization from both sarcolemma and t - tubules with similar kinetics .
a : t=0 shows confocal image of a basal glut4-egfp  expressing muscle fiber just before in situ contractions in an icr mouse .
contractions were elicited using the high - voltage protocol for 3  5 min separated by 90 sec of rest .
horizontal arrows indicate sarcolemma , while glut4-egfp depots are indicated near the nuclei by vertical arrows and inside the fiber by diagonal arrows .
t = denotes accumulated contraction time ; t = + denotes time during recovery after contractions .
b - d : image quantifications of glut4-egfp at the sarcolemma ( b ) , the t - tubules ( c ) , and the intracellular vesicle depots ( d ) .
ampk has been proposed to regulate contraction - stimulated glucose transport in skeletal muscle , although there is now considerable data suggesting that ampk is not necessary for this process ( 24,27,28 ) .
the role of ampk in regulating glut4 translocation kinetics in skeletal muscle has not been studied . here
, contraction - regulated glut4-egfp kinetics were compared in ampk2 inactive transgenic mice and their wild - type littermates . in the basal state
, there was a normal distribution of glut4-egfp in the intracellular depots and the perinuclear area ( fig .
3a and b ) ( t = 0 , diagonal and vertical arrow ) , with no continuous presence at the sarcolemma and no regular continuous striated pattern ( fig . 3a and b [ t = 0 ] ) .
thus , ablation of ampk2 activity did not affect glut4-egfp localization in the basal state .
ampk activity does not affect glut4-egfp basal localization and translocation and re - internalization kinetics in response to in situ contractions .
a and b : t = 0 shows confocal images of basal glut4-egfp  expressing muscle fibers immediately before in situ contractions in either a muscle - specific ampk2 inactive transgenic mouse ( a ) or a matched control mouse ( b ) .
contractions were elicited using the high - voltage protocol for 2  5 min separated by 90 sec of rest .
horizontal arrows indicate sarcolemma , while glut4-egfp depots are indicated near the nuclei by vertical arrows and inside the fibers by diagonal arrows .
numbers denote time in min during ( t =) and after ( t = + ) contractions .
c  e : image quantifications of glut4-egfp at the sarcolemma ( c ) , the t - tubules ( d ) , and the intracellular vesicle depots ( e ) .
ampk2 inactive mice and their matched controls were subjected to 2  5 min of high - voltage stimulation .
3a ) ( t = 0 , t = 10 , diagonal and vertical arrows ) to the sarcolemma ( fig .
3a ) ( t = 10 , horizontal arrow ) and t - tubules ( fig .
3a ) ( t = 10 , striated pattern ) . during 60 min of recovery ,
when compared with controls , there was no difference in glut4-egfp translocation and re - internalization ( fig .
image quantification showed that maximal translocation was reached at t = 10 min for both the sarcolemma ( 260% above basal ) ( fig .
this was accompanied by a 65% reduction in glut4-egfp , corresponding to 65% maximal reduction in nonperinuclear depots and a 55% maximal reduction in perinuclear glut4-egfp depots ( fig .
re - internalization of glut4-egfp occurred immediately after contractions with similar rates for the sarcolemma and t - tubules indicating similar kinetics ( fig .
ablation of ampk2 activity did not influence glut4-egfp kinetics in response to contractions ( fig .
e ) . thus , ampk2 activity is not necessary for normal glut4 trafficking kinetics before , during , and after in situ contractions in living animals .
a previous study suggested that the ampk activator aicar only increases glut4 translocation to the sarcolemma and not to the t - tubules ( 23 ) .
however , this finding is not consistent with the central role of the t - tubules in glut4 translocation ( 4,5,35 ) and glucose transport ( 15,35 ) and the finding that aicar stimulates glucose transport to the same extent as insulin and contraction ( 20 ) .
aicar was injected as a bolus into the tail vein immediately after t = 0 , and the first post - injection image was obtained 10 min later .
aicar resulted in a gradual but significant increase in staining along the sarcolemma by 2040 min after aicar injection ( fig .
furthermore , a clear striated pattern appeared by 2040 min after aicar injection demonstrating glut4-egfp translocation to the t - tubules ( fig . 4a [ t = 2040 ] and c [ supplementary video 2 ] ) .
maximal translocation occurred 90100 min after aicar injection with a 300% increase above basal for both the sarcolemma and t - tubules . increased glut4-egfp at the sarcolemma and t - tubules was accompanied by a gradual redistribution of glut4-egfp from depots ( fig .
the depot depletion corresponded to a 70% maximal reduction in nonperinuclear depots and a 60% maximal reduction in perinuclear glut4-egfp depots .
4 ) ( t = 20 , supplementary video 2 ) , and depletion further progressed at 80 min ( fig .
re - internalization of glut4-egfp from the sarcolemma and t - tubules followed an identical time course ( fig .
re - internalization kinetics were also similar in comparing the two stimuli ( figs . 1 , 2 , and 4 ) .
thus , aicar stimulates glut4-egfp translocation to both the sarcolemma and t - tubules in a similar manner to muscle contraction .
aicar stimulation induces glut4-egfp translocation to both sarcolemma and t - tubules with similar kinetics .
a : t = 0 shows a confocal image of a basal glut4-egfp  expressing muscle fiber in situ immediately prior to intravenous aicar administration via the tail vein of an anesthetized icr mouse .
horizontal arrows indicate sarcolemma , while glut4-egfp depots are indicated near the nuclei by vertical arrows and inside the fiber by diagonal arrows .
b  d : image quantifications of glut4-egfp at the sarcolemma ( b ) , the t - tubules ( c ) , and the intracellular vesicle depots ( d ) .
compared with control mice , the ampk2 inactive transgenic mice had minor staining along the sarcolemma ( fig .
5a and c ) and very weak striations in the t - tubule region following aicar injection ( fig .
furthermore , there was only limited depletion of glut4-egfp vesicle depots ( fig . 5b and e ) .
it is interesting that the changes in the glut4-egfp depots did not fully match sarcolemma and t - tubule glut4-egfp , as there was a modest depletion of glut4-egfp depots without an accompanying increase at the cell surface membranes ( fig .
, we believe that there is also an intermediate step in the glut4 translocation and re - internalization processes that appear as a light background  haze  upon imaging , and this haze can be observed in fig .
5a . depletion of ampk2 activity markedly reduces aicar - mediated glut4-egfp translocation to both the sarcolemma and t - tubules .
a and b : t = 0 shows confocal images of a basal glut4-egfp  expressing muscle fiber in situ in either an ampk2 inactive transgenic mouse ( a ) or a matched control fvb mouse ( b ) .
horizontal arrows indicate sarcolemma , while glut4-egfp depots are indicated near the nuclei by vertical arrows and inside the fiber by diagonal arrows .
c  e : image quantifications of glut4-egfp at the sarcolemma ( c ) , the t - tubules ( d ) , and the intracellular vesicle depots ( e ) .
the minimal glut4-egfp translocation that is present in these mice may be due to very low levels of ampk1 subunit in the muscle fibers and/or from nonspecific systemic effects of aicar .
in fact , we observed that injection of aicar caused minor trembling of the mice ( this can be seen as the muscle fiber vibrating in first half of supplementary video 2 ) .
thus , the majority of the aicar - induced glut4-egfp translocation is abolished in ampk2 inactive transgenic mice .
in the basal state , glut4-egfp was found in larger and smaller vesicle structures ( fig .
1d and e ) ( t = 0 , diagonal arrow ) and in the perinuclear region ( t = 0 , vertical arrow ) , as has been previously reported ( 4,5,16,29,36 ) .
1d and e ) ( t = 0 , horizontal arrow ) and no regular striated pattern throughout the fiber . following basal imaging ,
the superficial fibers of the quadriceps were contracted with high or low voltage . in mice subjected to high voltage contractions ,
there was marked staining along the sarcolemma by the 5 min time point ( fig .
1d ) ( t = 5 ) , quantified as a 60% increase above basal ( fig .
a striated pattern emerged inside the muscle fibers by 5 min showing translocation to the t - tubules ( fig .
, there was a 90% increase in t - tubule glut4-egfp after 5 min of contraction ( fig .
, there was a marked depletion of glut4-egfp vesicles in intracellular glut4-egfp vesicle depots , which also included the perinuclear region ( fig .
two additional 5-min contraction periods further increased glut4-egfp staining of sarcolemma and t - tubules ( > 200% increase from basal ) simultaneously with a further reduction in glut4-egfp vesicle depots ( 70% decrease from basal ) ( fig .
the depot depletion corresponded to a 75% maximal reduction in nonperinuclear depots and a 65% maximal reduction in perinuclear depots . with contractions elicited by low voltage
, there was a significant but modest glut4-egfp translocation to the sarcolemma by 10 min , which appeared in the form of continuous glut4-egfp staining ( fig .
the increases in glut4-egfp at the sarcolemma and t - tubules were accompanied by a 30% decrease in intracellular glut4 vesicle depots ( fig .
translocation to the sarcolemma and t - tubules steadily increased , and glut4-egfp vesicle depots were further reduced ( fig .
1e [ t = 20 , t = 30 , diagonal arrow ] and f  h ) .
1e ) ( t = 30 , horizontal arrow ) and t - tubules ( fig .
1e ) ( t = 30 , striated pattern ) occurred at the same time , indicating that glut4 translocation to these distinct cell surface membranes followed similar kinetics ( fig . 1f 
this is in contrast to what we have previously reported for insulin - mediated glut4-egfp translocation ( 5 ) .
comparison of high- and low - voltage contraction revealed that the high - voltage protocol resulted in greater levels of glut4-egfp translocation to the sarcolemma and t - tubules and a greater decrease in glut4 vesicle depots ( fig .
we next investigated the kinetics of glut4-egfp re - internalization in the period following muscle contractions , using the 3  5 min high - voltage contraction protocol that resulted in maximal translocation .
1 ) , glut4-egfp translocation to the sarcolemma and t - tubules increased gradually throughout the contractions , and there was a concomitant decrease in intracellular depots ( fig .
2 ) . interestingly , peak glut4-egfp depot depletion had occurred by 10 min ( fig .
2a [ t = 10 ] and d ) , whereas glut4-egfp translocation to the sarcolemma and t - tubules continued to increase during the first 10 min following the cessation of contractions ( fig .
this indicates a delay in the arrival of glut4-egfp at the sarcolemma and t - tubules .
following peak translocation to the sarcolemma and t - tubules , glut4-egfp underwent gradual net re - internalization and was almost fully re - internalized by 130 min after contractions ( fig .
the time course of glut4-egfp re - internalization was similar for the sarcolemma and t - tubules ( fig .
c ) . in situ contractions induce glut4-egfp translocation to and re - internalization from both sarcolemma and t - tubules with similar kinetics .
a : t=0 shows confocal image of a basal glut4-egfp  expressing muscle fiber just before in situ contractions in an icr mouse .
contractions were elicited using the high - voltage protocol for 3  5 min separated by 90 sec of rest .
horizontal arrows indicate sarcolemma , while glut4-egfp depots are indicated near the nuclei by vertical arrows and inside the fiber by diagonal arrows .
t = denotes accumulated contraction time ; t = + denotes time during recovery after contractions .
b - d : image quantifications of glut4-egfp at the sarcolemma ( b ) , the t - tubules ( c ) , and the intracellular vesicle depots ( d ) .
ampk has been proposed to regulate contraction - stimulated glucose transport in skeletal muscle , although there is now considerable data suggesting that ampk is not necessary for this process ( 24,27,28 ) .
the role of ampk in regulating glut4 translocation kinetics in skeletal muscle has not been studied . here ,
contraction - regulated glut4-egfp kinetics were compared in ampk2 inactive transgenic mice and their wild - type littermates . in the basal state
, there was a normal distribution of glut4-egfp in the intracellular depots and the perinuclear area ( fig .
3a and b ) ( t = 0 , diagonal and vertical arrow ) , with no continuous presence at the sarcolemma and no regular continuous striated pattern ( fig . 3a and b [ t = 0 ] ) .
thus , ablation of ampk2 activity did not affect glut4-egfp localization in the basal state .
ampk activity does not affect glut4-egfp basal localization and translocation and re - internalization kinetics in response to in situ contractions .
a and b : t = 0 shows confocal images of basal glut4-egfp  expressing muscle fibers immediately before in situ contractions in either a muscle - specific ampk2 inactive transgenic mouse ( a ) or a matched control mouse ( b ) .
contractions were elicited using the high - voltage protocol for 2  5 min separated by 90 sec of rest .
horizontal arrows indicate sarcolemma , while glut4-egfp depots are indicated near the nuclei by vertical arrows and inside the fibers by diagonal arrows .
numbers denote time in min during ( t =) and after ( t = + ) contractions .
c  e : image quantifications of glut4-egfp at the sarcolemma ( c ) , the t - tubules ( d ) , and the intracellular vesicle depots ( e ) .
ampk2 inactive mice and their matched controls were subjected to 2  5 min of high - voltage stimulation .
( t = 0 , t = 10 , diagonal and vertical arrows ) to the sarcolemma ( fig . 3a ) ( t = 10 , horizontal arrow ) and t - tubules ( fig .
3a ) ( t = 10 , striated pattern ) . during 60 min of recovery ,
when compared with controls , there was no difference in glut4-egfp translocation and re - internalization ( fig .
image quantification showed that maximal translocation was reached at t = 10 min for both the sarcolemma ( 260% above basal ) ( fig .
this was accompanied by a 65% reduction in glut4-egfp , corresponding to 65% maximal reduction in nonperinuclear depots and a 55% maximal reduction in perinuclear glut4-egfp depots ( fig .
re - internalization of glut4-egfp occurred immediately after contractions with similar rates for the sarcolemma and t - tubules indicating similar kinetics ( fig .
ablation of ampk2 activity did not influence glut4-egfp kinetics in response to contractions ( fig .
, ampk2 activity is not necessary for normal glut4 trafficking kinetics before , during , and after in situ contractions in living animals .
a previous study suggested that the ampk activator aicar only increases glut4 translocation to the sarcolemma and not to the t - tubules ( 23 ) .
however , this finding is not consistent with the central role of the t - tubules in glut4 translocation ( 4,5,35 ) and glucose transport ( 15,35 ) and the finding that aicar stimulates glucose transport to the same extent as insulin and contraction ( 20 ) .
aicar was injected as a bolus into the tail vein immediately after t = 0 , and the first post - injection image was obtained 10 min later .
aicar resulted in a gradual but significant increase in staining along the sarcolemma by 2040 min after aicar injection ( fig . 4a
furthermore , a clear striated pattern appeared by 2040 min after aicar injection demonstrating glut4-egfp translocation to the t - tubules ( fig . 4a [ t = 2040 ] and c [ supplementary video 2 ] ) .
maximal translocation occurred 90100 min after aicar injection with a 300% increase above basal for both the sarcolemma and t - tubules . increased glut4-egfp at the sarcolemma and t - tubules was accompanied by a gradual redistribution of glut4-egfp from depots ( fig .
the depot depletion corresponded to a 70% maximal reduction in nonperinuclear depots and a 60% maximal reduction in perinuclear glut4-egfp depots .
4 ) ( t = 20 , supplementary video 2 ) , and depletion further progressed at 80 min ( fig .
re - internalization of glut4-egfp from the sarcolemma and t - tubules followed an identical time course ( fig .
re - internalization kinetics were also similar in comparing the two stimuli ( figs . 1 , 2 , and 4 ) .
thus , aicar stimulates glut4-egfp translocation to both the sarcolemma and t - tubules in a similar manner to muscle contraction .
aicar stimulation induces glut4-egfp translocation to both sarcolemma and t - tubules with similar kinetics .
a : t = 0 shows a confocal image of a basal glut4-egfp  expressing muscle fiber in situ immediately prior to intravenous aicar administration via the tail vein of an anesthetized icr mouse .
horizontal arrows indicate sarcolemma , while glut4-egfp depots are indicated near the nuclei by vertical arrows and inside the fiber by diagonal arrows .
b  d : image quantifications of glut4-egfp at the sarcolemma ( b ) , the t - tubules ( c ) , and the intracellular vesicle depots ( d ) .
we determined if ampk2 was necessary for aicar - stimulated glut4-egfp translocation . compared with control mice , the ampk2 inactive transgenic mice had minor staining along the sarcolemma ( fig .
5a and c ) and very weak striations in the t - tubule region following aicar injection ( fig .
5b and d ) . furthermore , there was only limited depletion of glut4-egfp vesicle depots ( fig . 5b and e ) .
it is interesting that the changes in the glut4-egfp depots did not fully match sarcolemma and t - tubule glut4-egfp , as there was a modest depletion of glut4-egfp depots without an accompanying increase at the cell surface membranes ( fig .
, we believe that there is also an intermediate step in the glut4 translocation and re - internalization processes that appear as a light background  haze  upon imaging , and this haze can be observed in fig .
depletion of ampk2 activity markedly reduces aicar - mediated glut4-egfp translocation to both the sarcolemma and t - tubules .
a and b : t = 0 shows confocal images of a basal glut4-egfp  expressing muscle fiber in situ in either an ampk2 inactive transgenic mouse ( a ) or a matched control fvb mouse ( b ) . immediately after t = 0 , aicar was administrated intravenously via the tail vein .
horizontal arrows indicate sarcolemma , while glut4-egfp depots are indicated near the nuclei by vertical arrows and inside the fiber by diagonal arrows .
c  e : image quantifications of glut4-egfp at the sarcolemma ( c ) , the t - tubules ( d ) , and the intracellular vesicle depots ( e ) .
the minimal glut4-egfp translocation that is present in these mice may be due to very low levels of ampk1 subunit in the muscle fibers and/or from nonspecific systemic effects of aicar .
in fact , we observed that injection of aicar caused minor trembling of the mice ( this can be seen as the muscle fiber vibrating in first half of supplementary video 2 ) .
thus , the majority of the aicar - induced glut4-egfp translocation is abolished in ampk2 inactive transgenic mice .
physical exercise is accepted as an important modality to decrease blood glucose concentrations in patients with diabetes . despite the importance of exercise in the maintenance of overall glucose homeostasis , the cellular machinery that controls contraction - stimulated glucose transport
is poorly understood . in the current study , we used a newly developed imaging technique to investigate glut4 translocation by directly monitoring the kinetics of intramuscular glut4-egfp translocation before , during , and after contraction of skeletal muscle fibers in living mice .
we found that contraction increases glut4 translocation to both sarcolemma and t - tubules , this effect is voltage dependent , and glut4 on the cell surface membranes is increased for more than 2 h after the cessation of contraction . in examining a putative signaling mechanism , we found that ampk2 activity is not necessary for contraction - stimulated glut4 translocation .
translocation to the sarcolemma and t - tubules , as well as depletion of intracellular vesicle depots , was higher in response to contractions elicited by higher voltage despite a 50% shorter duration of contraction compared with the low - voltage protocol .
in addition , the high - voltage protocol resulted in a continuous increase in glut4-egfp translocation to the sarcolemma and t - tubules for the 15-min period even though maximal intracellular glut4-egfp vesicle depletion had occurred by 10 min ( fig .
the mechanism for the delay is not known ; however , it could be the result of the ability of high - voltage contraction to accelerate depletion of glut4-egfp storage vesicles rapidly , combined with a potential  bottleneck  at the exocytic / fusion step at the cell surface membranes .
such a bottleneck at the fusion step of glut4-egfp translocation has previously been shown to occur in primary adipocytes ( 10 ) .
the magnitude of the increase in glut4-egfp translocation was similar to that observed with myc - tagged - glut4 in sarcolemmal giant vesicles fractionated from contracted mouse muscle ( 37 ) and collagenase - derived cardiomyocytes ( 38 ) , although neither of these studies were performed in live animals .
current theory suggests that excitation - contraction coupling in the individual muscle fiber occurs as an all or nothing event ( 17 ) , and therefore an increase in stimulation above a threshold level would not have an additional effect on that muscle fiber .
in contrast , our observations , which followed glut4 translocation throughout the entire contraction treatment in the same muscle fibers , indicate that voltage - dependent regulatory mechanisms exist within individual fibers .
thus , metabolically derived signals may control the magnitude and duration of contraction - stimulated glut4 translocation as opposed to mechanically derived stimulation .
it is well established that there are distinct signaling mechanisms for exercise- and insulin - stimulated glucose transport , due in part to data demonstrating that the combination of a maximal insulin stimulus plus a maximal contraction stimulus has additive effects on glucose transport ( 19,39 ) and glut4 translocation ( 4 ) .
some studies suggest there are different intracellular pools of glut4 , one stimulated by insulin and one stimulated by exercise ( 18,40,41 ) .
although the current data do not directly support the possibility of different pools of glut4 in muscle , they do demonstrate that the spatial temporal kinetics of contraction- and insulin - stimulated glut4 translocation differ substantially . in a previous investigation , we analyzed recruitment , translocation , steady - state recycling , and subsequent re - internalization of glut4 in response to insulin in live mice . with insulin stimulation
, we found glut4-egfp translocation to the sarcolemma to be faster than glut4-egfp translocation in the t - tubules .
the delay was found to originate from a difference in initiation of the insulin signal . in parallel , glut4-egfp re - internalization
in contrast , in the present study , we found that the kinetics of contraction - stimulated glut4 translocation and re - internalization to and from the sarcolemma and t - tubules are similar for the two compartments .
these differences in glut4 translocation kinetics provide further evidence that the insulin- and contraction - stimulated translocation mechanisms for glut4 are distinct .
one of the reasons for this difference could be that the contraction impulses spread rapidly at a similar rate throughout the cell to both the t - tubules and sarcolemma during excitation - contraction coupling .
in contrast , insulin stimulation is initiated as insulin arrives first at the sarcolemma and , in turn , slowly diffuses into the t - tubules ( 5 ) .
another possible reason could be that the signaling / translocation cascade differs between the sarcolemma and t - tubules for insulin stimulation , whereas the contraction cascade does not .
in addition to contraction and insulin , there are many other stimuli that increase glucose transport in skeletal muscle ( 20 ) .
the ampk activator aicar increases glucose transport and , through the use of subcellular fractionation , we ( 36 ) and another group ( 23 ) have shown that aicar increases glut4 translocation . a more recent report ( 42 ) utilized a photolabeling technique and showed that aicar increased glut4 translocation to the membrane fraction of rat muscle strips to the same extent as a supraphysiological insulin dose .
surprisingly , in another report ( 23 ) , aicar was shown to only stimulate glut4 translocation to the sarcolemmal membranes and not to the t - tubules . in the current investigation ,
aicar clearly resulted in glut4 translocation to both the sarcolemma and t - tubules , and this redistribution of glut4 to the two membrane compartments adhered to similar kinetics .
the discrepancy may be due to very different experimental techniques with subcellular fractionation methods that are less sensitive .
given our current findings , the fact that aicar has robust effects on glucose transport and that detubulation experiments indicate a predominant role of t - tubules in regulating glucose transport in muscle ( 15,35 ) , we believe t - tubules play a significant role in the regulation of aicar - stimulated glucose transport .
ampk activation has been proposed to be the key regulator of contraction - stimulated glut4 translocation ( 43 ) , and ampk activation by aicar has been shown to increase glucose transport in skeletal muscle to the same extent as contractions ( 27 ) .
consistent with these findings , we observed that aicar and contractions resulted in similar increases in glut4 translocation in muscle fibers . to determine the role of ampk2 in aicar- and contraction - stimulated glut4 localization and trafficking , we studied ampk2 inactive transgenic mice .
aicar - stimulated glut4-egfp translocation was markedly reduced in the ampk2 inactive mice , consistent with previous results measuring abolished glucose transport ( 25,27 ) .
interestingly , contraction - stimulated glut4 translocation and re - internalization kinetics for both the sarcolemma and t - tubules were identical in mice with ablated ampk2 activity .
this is in line with a report that used subcellular fractionation to show that exercise in ampk2 kinase dead transgenic mice results in normal glut4 translocation ( 28 ) .
our glut4 translocation findings are consistent with studies showing only partial ( 25 ) or no ( 27 ) inhibition of glucose transport in models of ablated ampk2 .
thus , either ampk is not involved in contraction - induced glut4 translocation or redundant mechanisms mediate signaling to the intracellular glut4 vesicle depots .
interestingly , ablation of ampk2 activity had no effect on basal distribution of glut4 . in summary , this study determined the effects of contraction on glut4 translocation and re - internalization kinetics directly in the skeletal muscle of living mice . to describe glut4 translocation with muscle contraction ,
we and other groups have previously used a laborious subcellular fractionation methodology ( 44,45 ) .
this early work demonstrated that both insulin and exercise increase glucose uptake via glut4 translocation from an intracellular pool to the plasma membrane .
however , the novel microscopy technique described here has allowed us to significantly advance this line of investigation . by monitoring the localization and movement of fluorescent - labeled glut4 by confocal microscopy in the muscle fibers of anesthetized mice , we can observe the spatial - temporal details of glut4 localization and translocation .
previously , this level of quality and resolution of imaging was only possible in cell culture systems . in future studies ,
this method can be used to address important questions concerning contraction - induced glut4 translocation , such as how the glut4 vesicles interact with other proteins of the translocation machinery and the signals regulating glut4 translocation .
 objectiveexercise is an important strategy for the treatment of type 2 diabetes . 
 this is due in part to an increase in glucose transport that occurs in the working skeletal muscles . 
 glucose transport is regulated by glut4 translocation in muscle , but the molecular machinery mediating this process is poorly understood . 
 the purpose of this study was to 1 ) use a novel imaging system to elucidate the kinetics of contraction - induced glut4 translocation in skeletal muscle and 2 ) determine the function of amp - activated protein kinase 2 ( ampk2 ) in this process.research design and methodsconfocal imaging was used to visualize glut4-enhanced green fluorescent protein ( egfp ) in transfected quadriceps muscle fibers in living mice subjected to contractions or the ampk - activator aicar.resultscontraction increased glut4-egfp translocation from intracellular vesicle depots to both the sarcolemma and t - tubules with similar kinetics , although translocation was greater with contractions elicited by higher voltage . 
 re - internalization of glut4 did not begin until 10 min after contractions ceased and was not complete until 130 min after contractions . 
 aicar increased glut4-egfp translocation to both sarcolemma and t - tubules with similar kinetics . 
 ablation of ampk2 activity in ampk2 inactive transgenic mice did not change glut4-egfps basal localization , contraction - stimulated intracellular glut4-egfp vesicle depletion , translocation , or re - internalization , but diminished aicar - induced translocation.conclusionswe have developed a novel imaging system to study contraction - stimulated glut4 translocation in living mice . 
 contractions increase glut4 translocation to the sarcolemma and t - tubules with similar kinetics and do not require ampk2 activity . 
RESEARCH DESIGN AND METHODS
Plasmid and transfection procedures.
Time-lapse microscopy.
Image analysis.
Statistical analysis.
RESULTS
Contraction stimulates GLUT4-EGFP translocation to both the sarcolemma and t-tubules.
Kinetics of GLUT4-EGFP re-internalization.
Contraction-stimulated GLUT4-EGFP translocation and re-internalization are normal in AMPK2 inactive transgenic mice.
AICAR-stimulated GLUT4-EGFP translocation and re-internalization.
AICAR-stimulated GLUT4-EGFP translocation is impaired in AMPK2 inactive transgenic mice.
DISCUSSION
Supplementary Material