starvation and other altered metabolic conditions such as immobilization , denervation , 
 aging , and unloading states induces loss of muscle mass1,2,3,4 . to study the signal 
 transduction of atrophy in particular ,
various cell culture models have been developed5,6,7 . in many studies , exogenous cytokines such 
 as tnf-
, glucocorticoids such as dexamethasone , and serum - free starvation of cultured cells 
 have been used as atrophy models to confirm the mechanisms of whole skeletal muscle atrophy 
 in vivo8,9,10 .
the elevated degradation of proteins in 
 skeletal muscle atrophy and serum - free starvation is commonly coupled with activation of the 
 protein ligases such as muscle specific ring finger-1 ( murf-1 ) and atrogin-11 , 4 , 5 , 11
. 
 meanwhile , cofilin is a ubiquitously expressed protein in mammalian cells and thereby 
 regulates the actin filament dynamics and reorganization and other functions12,13,14 .
furthermore , cofilin binds to actin 
 molecules , changing fibrous actin to globular actin13 .
this process is enabled by the dephosphorylation of cofilin by 
 phosphatases12 , 15 . on the other hand , phosphorylation of cofilin
abolishes the 
 cofilin activity and inhibits its severing function12 , 16 ( fig . 1cfig .
1.change in phosphorylation of protein and schematic representation of the cellular 
 response caused by serum - free starvation with low glucose .
morphologic ( a - a30 , 
 a - b100 ) and immunoblotting ( b ) analyses in the starved l6 myoblasts .
fbs , fetal 
 bovine serum ; h , hours ; e. period , experimental period ; hg , high - concentration 
 glucose ; lg , low - concentration glucose ; p - cofilin , phosphorylated cofilin ; r , 
 receptor ; f - actin , fibrous actin ; g - actin , globular actin ; rho - rac - cdc42 , rho family 
 small gtpases ; rock , rho - associated protein kinase ; pak , p21-activated protein kinase ; 
 ssh , cofilin - specific phosphatases slingshot ; skeletal mcs , skeletal muscle cells . ) .
however , the changes in phosphorylation of cofilin in starvation - induced atrophy 
 are not fully understood .
therefore , we investigated the changes in the phosphorylation of 
 cofilin in l6 myoblasts during serum - free starvation with low glucose
. change in phosphorylation of protein and schematic representation of the cellular 
 response caused by serum - free starvation with low glucose .
morphologic ( a - a30 , 
 a - b100 ) and immunoblotting ( b ) analyses in the starved l6 myoblasts .
fbs , fetal 
 bovine serum ; h , hours ; e. period , experimental period ; hg , high - concentration 
 glucose ; lg , low - concentration glucose ; p - cofilin , phosphorylated cofilin ; r , 
 receptor ; f - actin , fibrous actin ; g - actin , globular actin ; rho - rac - cdc42 , rho family 
 small gtpases ; rock , rho - associated protein kinase ; pak , p21-activated protein kinase ; 
 ssh , cofilin - specific phosphatases slingshot ; skeletal mcs , skeletal muscle cells .
l6 myoblasts from rat neonate skeletal muscle were separated into control and serum - free 
 starvation groups1 .
the control group of 
 l6 myoblasts was purchased from the american type culture collection ( rockville , md , usa ) 
 and cultured in dulbecco s modified eagle s medium containing 10% fetal bovine serum , 100 
 u / ml penicillin , 100  g / ml streptomycin , 200  mm glutamine , and 4,500  mg / l high - concentration 
 d - glucose .
the serum - free starvation group of l6 myoblasts grown to 6070% confluence and 
 undernourished in dmem containing 1,000  mg / l low - concentration d - glucose without fbs for 3 , 
 6 , 12 , 24 , 48 , and 72 h , respectively1 . 

after each experimental treatment , cells were lysed with an extraction buffer ( 20  mm hepes , 
 ph 7.5 , 1% nonidet p-40 , 150  mm nacl , 10% glycerol , 10  mm naf , 1  mm 
 na3vo4 , 2.5  mm 4-nitrophenylphosphate , 0.5  mm pmsf , and one tablet 
 of complete proteinase inhibitor cocktail [ roche , indianapolis , in , usa ] ) .
the morphological 
 changes in l6 myoblasts with or without each experimental treatment were visualized with an 
 inverted microscope ( ae30/31 , motic incorporation , richmond , bc , canada ) . to measure the 
 phosphorylation of cofilin
proteins ( 3045 g / lane ) were 
 separated on 12% polyacrylamide sodium dodecyl sulfate gels and then transferred 
 electrophoretically to a polyvinylidene fluoride membrane ( millipore ; bedford , ma , usa)2 .
anti - cofilin antibody was purchased from 
 santa cruz biotechnology ( santa cruz , ca , usa ) .
antibody - specific bands were quantified 
 using an image analyzer ( bio - rad ) .
the protocol for the study was approved by the committee 
 of ethics in research of the university of yongin , in accordance with the terms of 
 resolution 5 - 1 - 20 , december 2006 .
the data were 
 statistically evaluated using student s t - tests for comparisons between pairs of groups and 
 by anova for multiple comparisons . a p value of < 0.05 was considered to be statistically 
 significant .
l6 cell sizes and numbers were diminished as a result of serum - free starvation in a 
 time - dependent manner ( fig .
phosphorylation 
 of cofilin was significantly decreased after 3 , 6 , 12 , 24 , 48 , and 72 hours of starvation 
 compared with those of the control groups ( n=34 , fig . 
 1b , table 1table 1.changes in expression and phosphorylation of cofilin of l6 myoblasts during 
 serum - free starvation with low glucoseexperimental periodcofilin ( % ) p - cofilin ( % ) 0 hour ( control)100.00.0100.00.03 hours252.329.5 * 20.76.1 * 6 hours242.720.7 * 25.34.6 * 12 hours201.026.2 * 24.76.3 * 24 hours198.722.9 * 26.35.9
* 48 hours196.022.1 * 24.35.4 * 72 hours176.314.4 * 18.05.5*data were presented as the mean  sem .
the basal levels of 
 abundance and phosphorylation in controls ( 0 hour ) were considered to be 100% . 
 * compared with the 0 hour control , p<0.05 . ) .
however , the expression of cofilin was significantly increased after 3 , 6 , 
 12 , 24 , 48 , and 72 hours of starvation compared with the expression of cofilin in the 
 control groups ( n=34 , fig .
p , phosphorylated protein . the basal levels of 
 abundance and phosphorylation in controls ( 0 hour ) were considered to be 100% . 
 * compared with the 0 hour control , p<0.05 .
skeletal muscle atrophy and joint contracture have proven to be significant orthopaedic 
 problems in the area of physical therapy3 , 17 , 18 .
the skeletal muscle is the largest organ with high plasticity in the 
 human , comprising about 50% of the total body weight .
maintenance of muscle mass and 
 neuromuscular function is important for activities daily living ( adl ) in patients1 , 2 , 19 , and maintenance of muscle mass in 
 particular is related in part to optimal nutrient absorption and use . in contrast , nutrient 
 starvation and disuse have in part the potential to negatively impact muscle mass such as 
 through skeletal muscle atrophy20,21,22,23 .
our previous study reported that the 
 ubiquitin protein ligases murf-11 and 
 atrogin-1 ( data not shown ) , markers of muscle atrophy , are increased in atrophied 
 gastrocnemius muscle strips and involved in the development of serum - free starvation - induced 
 atrophy in l6 myoblasts . simultaneously , extracellular signal - regulated kinase 1/2 ( erk1/2 ) , 
 stress - activated protein kinase / c - jun nh2-terminal kinase 
 ( sapk / jnk ) , and p38 mitogen - activated protein kinase ( p38mapk ) are involved in atrophy 
 caused by cast immobilization of a hind limb and serum - free starvation of l6 myoblasts1 , 2 . 

also , our previous report demonstrated that cast immobilization of rat gastrocnemius muscles 
 increases the expression of tissue myoglobin24 .
meanwhile , cofilin in eukaryotic cells binds to actin and plays a 
 role in actin dynamics and reorganization involved in cast immobilization - induced 
 atrophy14 , 25 .
phosphorylation of cofilin is achieved by lim kinases and thereby 
 inhibits the actin binding , severing , and depolymerizing activities of cofilin16 , 25 
 ( fig .
furthermore , the kinases responsible 
 for this phosphorylation are rho - associated protein kinase and p21-activated protein kinase , 
 which are downstream kinases of the rho family small gtpases26,27,28 . on the other hand , dephosphorylation of cofilin
is mediated by the 
 cofilin - specific phosphatases slingshot12 , 15 ( fig . 

although cofilin is essential for maintenance of skeletal muscle mass12 , it has not been reported that 
 phosphorylation of cofilin is related to atrophy caused by serum - free starvation in the area 
 of physical therapy . however , further systematic studies in the area of physical therapy 
 such as electrotherapy , neurotherapy , hydrotherapy , and others are needed to confirm the 
 mechanism of cofilin under atrophic conditions29,30,31 ( fig .
in summary , 
 the phosphorylation of cofilin was decreased in starved skeletal muscle cells .
the present 
 results suggest that serum - free starvation - induced atrophy may be in part mediated by 
 cofilin from l6 myoblasts .
 [ purpose ] many studies have been using cell culture models of muscle cells with exogenous 
 cytokines or glucocorticoids to mimic atrophy in in vivo and in vitro tests . 
 however , the 
 changes in the phosphorylation of atrophy - related cofilin are still poorly understood in 
 starved skeletal muscle cells . in this study 
 , we first examined whether or not 
 phosphorylation of cofilin is altered in l6 myoblasts after 3 , 6 , 12 , 24 , 48 , and 72 hours 
 of serum - free starvation with low glucose . [ methods ] we used western blotting to exam 
 protein expression and phosphorylation in atrophied l6 myoblasts . 
 [ results ] l6 cell sizes 
 and numbers were diminished as a result of serum - free starvation in a time - dependent 
 manner . 
 serum - free starvation for 3 , 6 , 12 , 24 , 48 , and 72 hours significantly decreased 
 the phosphorylation of cofilin , respectively . 
 [ conclusion ] these results suggest that 
 starvation - induced atrophy may be in part related to changes in the phosphorylation of 
 cofilin in l6 myoblasts . 