Paternal Treadmill Exercise Affects Skeletal Muscle MTOR Signaling Pathway In The Offspring Of C57BL/6Mice

Objective:Skeletal muscle, accounting for over40%of the total body weight, is a highly plastic tissue and plays a crucial role in whole body metabolism. Developing research has demonstrated that many of the benefits of exercise are mediated through the role of skeletal muscle as an endocrine organ. Evidence have indicated skeletal muscle mass is controlled by the balance between protein synthesis and protein degradation. To date, the specific regulation mechanism is not fully clear. Highly conservative mammalian target of rapamycin (mTOR) signaling pathway has been proposed to play a key role in the process of skeletal muscle hypertrophy.Epidemiological studies have suggested the important role of parental lifetime exposures to environmental challenges in providing a trajectory for health in the offspring. Three weeks old mice born to mothers who exercised regularly during pregnancy had improved glucose homeostasis and decreased fat mass. Animal studies also demonstrated that pregnant rats with exercise had lower fetal birth weight, male offspring from exercised mice showed increased percent lean mass compared with male offspring from sedentary mice. Recently study also showed exercise during pregnancy improved offspring insulin sensitivity and glucose homeostasis. To date, studies examining the effects of maternal exercise on fetal growth have focused primarily on offspring birth size, with inconsistent findings. However, knowledge about the contributions of paternal aerobic exercise before mating and the long-term consequences on offspring is minimal. It is therefore of great interest to determine whether paternal aerobic exercise have effects in mammals and to characterize the mechanisms that mediate these effects. Thus, we assessed skeletal muscle mass, the grip strength, the quadriceps femoris muscle cross-section area and the activity of mTOR signaling pathway in the offspring.Methods:(1) Animal model:Sibling males were divided into two groups:the control (C) and the exercise group (E). Sibling females were kept sedentary. The mice in the running group were exercised on a motor-driven rodent treadmill for5days a week for a total of6weeks. After6weeks of exercise, one male and one female were mated. To minimize male-female interactions, which may impact on maternal investment or care and since affect offspring development, pregnancy was assured by observing a copulation plug (identified each morning within1h after lights on) and signaled to remove the female to her own cage. F1male mice of exercised group and sedentary group were named for EM and CM, F1female mice of exercised group and sedentary group were named for EF and CF specially;(2) Exercise training protocol: E group were given exercise training on motorized treadmill at12m/min (75%VO2max) for60min/day, once per day for6weeks (5days per week);(3) Grip testing: After weaning (21days of age), the weight and the forelimb grip strength of each mouse were recorded. Each mouse was allowed to grab a bar attached to a force transducer as it was pulled by the tail horizontally away from the bar. Five repetitions with a5s pause between each were averaged to determine grip strength for each mouse;(4) H&E staining:The H&E staining is a classic nucleic acid staining method traditionally used on tissue observe in morphology. All pups were weighed and killed the morning following the last treatment day and were fasted overnight (12h) with free access to water. Quadriceps femoris muscle were removed from euthanized mice and fixed in10%of neutral buffered formalin, embedded in paraffin. The paraffin was blocked6mm sections and stained with hematoxylin and eosin and then photographed at400×magnifications. The muscle cross sectional area was calculated by using Image J software;(5) Real-time PCR was used to detect PI3K, Akt, mTOR, S6K1and4E-BP1mRNA levels in quadriceps femoris muscle of EM and CM mice;(6) Western blot was used to detect PI3K, Akt, mTOR and S6K1total protein expression levels in quadriceps femoris muscle of EM and CM mice;(7) Western blot was used to detect Akt, mTOR, S6K1and4E-BP1phosphorylation protein levels in quadriceps femoris muscle of EM and CM mice.Results:(1) Morphological characteristics (body weight, quadriceps femoris muscle wet weight and quadriceps femoris muscle wet weight/body weight):Compared to CM group, the body weight, quadriceps femoris muscle wet weight and the ratio of quadriceps femoris muscle wet weight/body weight were significantly increased by12.76%,23.50%and9.73%in EM pups (P<0.05) However, we found no significant difference in female offspring (data not shown);(2) Grip testing:The results of the grip test showed that the ratio of grip strength/body weight in EM pups was increased by21.50%(P<0.05) comparing to CM group; Likely, we also found no significant difference in female offspring (data not shown); Therefore, we only detected CM and EM group mice in the following study;(3) H&E staining demonstrated that paternal exercise increased quadriceps femoris muscle mean cross sectional area by30.26%(P<0.05) and increased the percentage of large fibers, reduced the percentage of small fibers of EM group compared with CM group;(4) PI3K, Akt, mTOR, S6K1and4E-BP1mRNA levels in quadriceps femoris muscle of male offspring:Compared with CM group, PI3K, Akt, mTOR, S6K1and4E-BP1mRNA levels increased16.87%,10.96%,22.48%,83.12%and73.26%respectively in EM group, but only S6K1and4E-BP1mRNA expression increased significantly;(5) PI3K, Akt, mTOR and S6K1total protein levels in quadriceps femoris muscle of male offspring: Total protein expression levels of PI3K, Akt, mTOR and S6K1in mice of EM group were increased comparing to CM group, but had no statistical significance;(6) Akt, mTOR, S6K1and4E-BP1phosphorylation protein levels in quadriceps femoris muscle of male offspring:Compared with CM group, pAkt, pmTOR, pS6Kl and p4E-BPl protein levels were significantly increased by14.63%,14.83%,92.74%and74.11%respectively in EM group (P<0.05).Conclusions:In our present study, we proposed that paternal treadmill exercise enhanced quadriceps femoris muscle growth, increased quadriceps femoris muscle mean cross sectional area, stimulated muscle hypertrophy and thereby increased muscle grip strength of male pups. This is the first study examining the effects of paternal regular aerobic exercise on skeletal muscle growth of offsprings. We also have investigated the possibility that this positive effect of paternal treadmill exercise on skeletal muscle growth of male pups might be partially mediated by exercise-induced activation of mTOR signaling pathway in quadriceps femoris muscle. But further studies are needed to explore why we did not find these changes in female offsprings and elucidate the underlying mechanisms by which mTOR signaling pathway mediated the paternal exercise-induced improvement of skeletal muscle functions of male pups.