obesity increases the risk of metabolic disorders , such as high blood pressure , type 2 
 diabetes , and fatty liver1,2,3 .
it can also reduce 
 skeletal muscle mass and exert a negative effect on normal muscle function4 , 5 .
mammalian target of rapamycin ( mtor ) , a serine / threonine protein kinase regulating the 
 functions and activities of various cells in the skeletal muscle , consists of 2 distinct 
 protein complexes :  mtorc1 and  mtorc26 . 
mtorc1 , a master regulator of protein synthesis and cell growth , 
 contains the scaffold protein raptor , whereas glucose  mtorc2 , a regulator or akt activity , 
 contains rictor .
akt , which 
 transduces important signals for regulating cell metabolism and cell growth , is activated by 
 rictor .
the activated akt , in turn , induces protein synthesis and muscle fiber hypertrophy 
 via the s6 kinase 1 ( s6k1 ) pathway9,10,11 . 
 however , the overactivation of the raptor - s6k1 signaling pathway causes obesity by promoting 
 fat deposition in the muscle , liver , and white adipose tissue12 . in spite of the raptor - s6k1 activity , an over - nutrition condition 
 such as obesity leads to muscle wasting and functional damage because of the low efficacy of 
 protein anabolism13 .
the reduction in 
 rictor - akt activity causes a reduction in muscle mass by promoting protein catabolism7 ; and further , it causes metabolic imbalance 
 leading to increase in oxidative stress , inflammatory responses , and the risk of 
 cardiovascular diseases and obesity12 .
many studies have reported that both acute and regular resistance exercises promoted 
 protein synthesis and muscle hypertrophy by activating the  mtor pathway14,15,16 . yet
, there is very limited research that investigated the 
 change in the  mtor signaling pathway according to regular aerobic exercise , though aerobic 
 exercise is the most effective type of exercise for treating obesity .
moreover , it is 
 notable that aerobic excise can inhibit  mtorc1 by inducing adenosine monophosphate - activated 
 protein kinase ( ampk ) activity that promotes oxidative metabolism17 , 18 .
this suggests 
 that aerobic exercise can reduce muscle hypertrophy , and therefore , induce a different 
 pattern of change in the  mtor signaling pathway compared to resistance exercise .
thus , although exercise is an important factor that induces muscle protein synthesis and 
 muscle hypertrophy by enhancing the positive effect of the  mtor signaling pathway , most of 
 the preceding researches have been limited to resistance exercise .
in particular , there has 
 been insufficient research that involves obesity models to investigate the effect of regular 
 aerobic exercise training on the rictor - akt and  mtor - raptor - s6k1 signaling pathways .
therefore , the present research studied the effect of 8 weeks of regular treadmill exercise 
 on the skeletal muscle rictor - akt and  mtor - raptor - s6k1 signaling pathways in high - fat 
 diet - induced obese mice .
forty male c57bl/6 mice ( 4 weeks old ) were housed in cages and fed freely with standard rat 
 chow and water ( samtako , inc .
four mice were housed in each cage and maintained 
 under standardized conditions in an animal facility ( laboratory of animals , college of 
 medicine , dong - a university ) , at a room temperature of 22  2  c , 60  5% relative humidity , 
 and a 12  h light / dark cycle .
animal experiments were approved by dong - a university medical 
 school institutional animal care and use committee ( diacuc-1321 ) , and all procedures were 
 conducted in accordance with committee guidelines .
after 1 week of adaptation maintenance , mice were randomly divided into2 groups to induce 
 obesity by high - fat diet for 8 weeks : a normal diet ( nd ) group ( 6% fat ; samtako , inc .
, 
 korea , n = 20 ) , and a high fat diet ( hf ) group ( 45% fat ; research diets inc .
dietary and body weight were measured every week at the same time ( 11:00 ) during the entire 
 experimental period . after inducing obesity ,
mice were randomly subdivided into the nd , nd , and training ( ndt ) , 
 hf , hf , and training ( hft ) groups ( 10 mice per group ) .
exercise protocol was adapted for 5 
 days in order to perform the treadmill exercise .
exercise for mice in the training groups 
 was conducted on a treadmill for 40  min once a day , 5 times a week , for 8 weeks .
exercise 
 intensity consisted of 5 m / min ( 5  min ) , 10 m / min ( 30  min ) , and 5 m / min ( 5  min ) at 0% slope 
 for weeks 1 to 4 ( low intensity ) . during weeks 5 to 8
, exercise intensity was increased to 5 
 m / min ( 5  min ) , 14 m / min ( 30  min ) , and 5 m / min ( 5  min ) at the same slope ( moderate 
 intensity ) .
tissues were collected at 48  h after the completion of training in order to rule out 
 temporary exercise effects .
the feed supply was discontinued 12  h prior to sampling with 
 water continuously supplied .
muscles were 
 removed to collect the soleus muscle ( 10  g ) and stored in a deep freezer ( nihow freezer , 
 japan ) at 80  c until analysis . to extract protein from the soleus muscle ,
tissues were crushed after adding a solution 
 containing 150  mm nacl , 5  mm edta , 50  mm tri - hcl ( ph 8.0 ) , 1%-np 40 , 1  mm aprotinin , 0.1  mm 
 leupeptin , and 1  mm pepstatin .
the solution was centrifuged for 30  min at 16,600  g. 
 supernatants were collected and assayed for protein content prior to storage at 80  c . 

protein samples were mixed with laemmli sample buffer ( lsb ) and placed in a boiling water 
 bath for 5  min .
proteins were resolved by 10 , 12 , or 15% sds - polyacrylamide gel 
 electrophoresis ( sds - page ; each loaded with same amount in g of total protein per lane ) , 
 and transferred to nitrocellulose membranes .
thereafter , protein membranes were incubated 
 with the following primary antibodies : akt ( 1:1000 ; cell signaling ) ,  mtor ( 1:1000 ; cell 
 signaling ) , raptor ( 1:1000 ; cell signaling ) , rictor ( 1:1000 ; cell signaling ) and s6k1 
 ( 1:1000 ; cell signaling ) for 1 hour , and washed 3 times ( 15  min each ) in a pbs solution 
 containing 0.1% tween 20 .
the washed membrane was then treated with secondary antibody 
 ( goat - anti - mouse [ or rabbit ] igg ) conjugated with horseradish peroxidase ( hrp ) . 

the relative strengths of bands were 
 quantitated by imagequanttm las-4000 ( ge healthcare , swe ) .
all data were analyzed using spss software version 21.0 for windows ( spss inc . ,
body weight and  mtor signaling 
 pathway ( mtor , raptor , s6k1 , rictor , akt ) were analyzed by one - way anova .
the body weight of mice at 8 weeks of exercise training was significantly higher in the hf 
 ( 49.1  0.8  g ) group than that in the nd ( 38.1  1.5  g ) and ndt ( 31.7  1.3 g ) groups ( p 
 < 0.05 ) .
in addition , body weight in the hft ( 42.4  1.1  g ) group was significantly lower 
 than that in the hf group ( p < 0.05 ) ; body weight in the ndt group was also significantly 
 different from that in the nd and hft groups ( p < 0.05 ) . the  mtor , raptor / mtorc1 , s6k1 , rictor / mtorc2 , and akt levels for the 4 groups after 8 weeks 
 of exercise training are shown in table 
 1table 1.changes in mtor signaling pathway factors in skeletal muscle after 8 weeks of exercise training . 
mtor levels were significantly higher in the hf group than that in the nd and 
 ndt groups ( p < 0.05 ) .
raptor / mtorc1 and s6k1 levels were significantly higher in the hf 
 group than in all the other groups ( p < 0.05 ) .
akt levels were significantly lower in the 
 hf group than that in the ndt group ( p < 0.05 ) .
in contrast , rictor / mtorc2 levels were 
 not significantly different among all the groups .
an obese individual may suffer deterioration of muscle function owing to the reduction of 
 muscle mass caused by imbalance in protein synthesis and degradation19 .
it is , therefore , important to maintain or increase 
 skeletal muscle mass through mechanical stimulus such as exercise .
russell20 reported that skeletal muscle mass is 
 regulated by the synergy between the growth and loss of muscle and is closely associated 
 with the  mtor signaling pathway .
mtor has been reported to be regulated by repetitive muscle contraction and , as a mediator 
 of the adaptation induced by exercise , to promote the growth and hypertrophy of muscle fiber 
 by being involved in protein synthesis21 . 

richard - bulteau et al.22 reported 
 that  mtor expression decreased in damaged skeletal muscle but increased during the initial 
 period of exercise , and that  mtor played an important role in muscle growth .
in the present 
 study , the hf group showed significantly higher  mtor levels compared to the nd and ndt 
 groups .
this result was because of high - fat diet - induced obesity , which suggests that 
 regular aerobic exercise could not induce  mtor activity . of interest ,
li et al.23 reported the tendency of the  mtor 
 signaling pathway to be overactivated in the skeletal muscle of obese individuals .
our 
 results are also supported by those of mller et al.24 , who reported that resistance exercise could induce  mtorc1 
 activation in skeletal muscle but endurance exercise exerted no significant effect .
it has been reported that raptor , a component of  mtor complex , plays an important role in 
 regulating protein synthesis , mitochondrial biosynthesis , and oxidative metabolism in 
 muscle7 , and that repetitive muscle 
 contractions could induce skeletal muscle growth by increasing raptor activity25 . on the contrary ,
detective raptor in 
 muscle could reduce oxidative metabolism in mitochondria and cause early death26 . in the present study ,
the hf group showed 
 a significantly higher raptor levels compared to the other 3 groups ( the nd , ndt , and hft 
 groups ) .
this result is in accordance with those of preceding researches which showed 
 significantly higher level of raptor in the muscles of high fat - fed obese rats27 , which proved that aerobic exercise could 
 decrease raptor activity18
. it can be 
 interpreted from our results that raptor activity was abnormally overactivated by high - fat 
 diet - induced obesity rather than by skeletal muscle synthesis , but that aerobic exercise 
 positively contributed to normalizing the raptor signaling pathway .
this interpretation is 
 supported by the suggestion that chronic raptor activation or abnormal raptor signaling 
 might rather weaken the skeletal muscle response to growth signals in obesity models28 , 29 
 and also by the suggestion from laplante and sabatini7 that no increase of muscle mass , in spite of high level of raptor 
 activity in obesity models , is because of protein catabolism .
s6k1 , which is involved in the mechanism of raptor downstream signaling , integrates various 
 external signals for controlling cell growth and metabolism to induce protein synthesis and 
 muscle growth30 .
pagano et al.31 reported an increase in s6k1 during 
 recovery after moderate- or high - intensity endurance exercise . in the present study ,
the hf 
 group showed a significantly higher s6k1 levels than the other 3 groups , similar to that of 
 raptor .
considering the high level of raptor shown by the hf group , it can be interpreted 
 from our results that obesity is the main contributor to s6k1 activity , but that aerobic 
 exercise suppressed the obesity - induced overactivation of s6k1 .
this interpretation is 
 supported by previous studies , which reported that the activation of the raptor - s6k1 
 signaling pathway was associated not only with protein synthesis but also with obesity32 , 33 .
liao and xu32 , 
 who investigated  mtor / s6k1 signaling in high - fat diet - induced obese rats , also demonstrated 
 the remarkable increase of s6k1 in their muscles .
in addition , khamzina et al.27 suggested that high - fat diet - induced 
 increase in  mtorc1 activity is because of increased s6k1 phosphorylation , whereas williamson 
 et al.18 suggested that the decrease in 
 raptor activity caused by aerobic exercise is because of the decrease in s6k1 .
rictor plays an important role in maintaining the life and size of cells by being involved 
 in the long - term activation of akt / protein kinase b34 , 35 , and its interaction with 
 akt is associated with the regulation of carbohydrate metabolism in cells36 .
the role of exercise - induced rictor 
 activation is not yet clearly known , though it has been reported that aerobic 
 exercise - induced  mtorc2 activity promoted metabolism8 . in the present study , while there was no significant difference in 
 rictor , akt level was found to be significantly lower in the hf group than in the ndt group . 

preceding studies have reported that akt was closely associated with the regulation of  mtor 
 and s6k1 levels , and that increase in akt expression contributed to improving high - fat 
 diet - induced obesity37 , 38 and increased anabolism in muscles39 .
it can be interpreted from our results that akt 
 expression was suppressed by obesity in the hf group , but that akt expression increased in 
 the ndt group as an efficacy of regular aerobic exercise .
altogether , the results suggest that risk of obesity may be associated with the 
 overactivation of the  mtor - raptor - s6k1 signaling pathway and a decrease in akt levels
. we 
 also found that performing aerobic exercise may be associated with the downregulation of 
 the  mtor - raptor - s6k1 pathway and an increase in akt levels . whereas a reduction in the 
 concentration of proteins related to  mtor signaling according to aerobic exercise may not 
 reflect its negative effects on metabolism and protein synthesis in the skeletal muscle
 [ purpose ] the aim of this study was to investigate the effects of regular treadmill 
 exercise on skeletal muscle rictor - akt and  mtor - raptor - s6k1 signaling pathway in high - fat 
 diet - induced obese mice . [ subjects and methods ] four- week - old c57bl/6 mice were adopted 
 and classified into normal diet group ( nd , n = 10 ) , normal diet and training group ( ndt , n 
 = 10 ) , high - fat diet group ( hf , n = 10 ) , and high - fat diet and training group ( hft , n = 
 10 ) . the exercise program consisted of a treadmill exercise provided at low intensity for 
 14 weeks , and moderate intensity for 58 weeks . [ results ] 
 the western blot method was 
 used to measure the expression of  mtor , raptor , s6k1 , rictor , and akt proteins in the 
 soleus muscle .  
 mtor levels were significantly higher in the hf group than in the nd and 
 ndt groups . 
 raptor / mtorc1 and s6k1 levels were significantly higher in the hf group than 
 in all the other groups . 
 akt levels were significantly lower in the hf group than in the 
 ndt group . 
 the risk of obesity may be associated with the overactivation of 
 the  mtor - raptor - s6k1 signaling pathway and a decrease in akt levels . 
 [ conclusion ] this 
 study also indicates that performing aerobic exercise may be associated with the 
 downregulation of the  mtor - raptor - s6k1 pathway . 
INTRODUCTION
SUBJECTS AND METHODS
RESULTS
DISCUSSION