The Effects Of Exercise On Mitochondrial Biogenesis And The Underlying Signaling Transduction Mechanisms

Skeletal muscle exhibits remarkable adaptive capabilities in response to a number of physiological and pathophysiological conditions.Chronic contractile activities induce mitochondrial biogenesis and increase fatty acids oxidation in skeletal muscle.These adaptations are highly specific and dependent upon the type of exercise(i.e.)resistance vs endurance)as well as its frequency,intensity and duration.A coordinated alteration in the expression of a variety of nuclear DNA and mitochondrial DNA(mtDNA)gene is needed for the adaptations.The molecular mechanism still remains unclear.One of the most important biological processes is ROS production in the exercise-induced mitochondrial biogenesis which changes the redox state of cell.Acute exercise may be a stress for the body because of the increased ROS production.The ROS induced by long term moderate exercise may play critical role in mitochondrial biogenesis.Studies suggest mTOR signal is associated with mitochondrial function and is regulated by the redox state of cell.This may be the potential mechanism links mitochondrial activity to mTOR under regulation of nutrition and other physiological and pathophysiological conditions.The effect of exercise on mTOR signal is not clear.It is thought that endurance exercise and resistance exercise can selectively activate either AMPK/PGC-1αor Akt/mTOR signaling,respectively and the activated AMPK/PGC-1αmay inhibit Akt/mTOR signaling.However,there are some contradict findings.More delicately designed study is needed to make clear about the effects of exercise on AMPK/PGC-1αand Akt/mTOR signaling and it is worth to probe the role of AMPK/PGC-1αas well as Akt/mTOR signal pathway in mitochondrial biogenesis and metabolism.There is a growing understanding that gene-specific transcriptional activation during and after a bout of exercise is involved,to some extent,in reestablishing homeostasis in skeletal muscle after the exercise bout,as well as contributing to the skeletal muscle adaptations that occur in response to exercise training.PGC-1αis thought to play a pivotal role in these processes.The positive effects of PGC-1αon transcription are thought to be mediated by its ability to bind to specific transcription factors recruit additional coactivators,thereby enhancing the overall efficiency of the transcriptional machinery.PGC-1αcan influence fatty acids oxidation andβ-oxidation through co- activating PPARαandγand influence uncoupling respiration by regulating UCPs transcription.NRF-1,as a strong coactivator of PGC-1α,leads to increased levels of several nuclear-encoded mitochondrial proteins,including Tfam,provokes an increase in mtDNA transcription and replication.Tfam,together with TFB1M and TFB2M,which is closely associated with NRFs,is responsible for the expression of various transcription factors coded by nuclear DNA and mtDNA.Although the entire transcriptional response to endurance exercise has not been currently demonstrated,a single bout of endurance exercise has been shown to increase mRNA expression of a growing number of genes, the majority of which are involved in mitochondrial biogenesis and fatty acids metabolism,such as NRFs,Tfam,PPARα,PDK4,LPL and CPTâ… .The purposes of the current study were as follows.One was to test the effects of different intensities of exercise,which included moderate intensity and high intensity,on mitochondrial biogenesis and to test the role of mTOR signal in the exercise-induced mitochondrial biogenesis.The second purpose was to test the effects of two kinds of acute cycling exercise on the expression of various transcription factors which related to mitochondrial biogenesis and fatty acids oxidation.1 The effects of exercise and exercise combined with mTOR inhibitor on rat mitochondrial biogenesis.Method:Following one week of adaptive feeding,24 SD rats underwent adaptive training for 3 days.After this,they were divided into different groups.The first 18 rats were divided into 3 groups which included control group(C),moderate intensity exercise group(M)and high intensity exercise group(H).The second 24 rats were divided into 4 groups which include control(C),RPM group(R),moderate intensity exercise group(M) and moderate intensity exercise combined with RPM group(MR).There are 6 rats in each group.For the first 18 rats,following 1 week of progressed increasing intensity exercise,M and H groups were trained on the treadmill under moderate and high intensity for 1 hour every time and 5 times a week which last for 2 weeks and C group didn't do any exercise. They were killed 48 hours after the last time exercise.Mitochondria were isolated from skeletal muscle and were used for testing ROS production.The other part of skeletal muscle were saved in -80℃for testing complexâ… ,â…¡andâ…£proteins level as well as mtDNA and COXâ…£gene expression later.For the second 24 rats,following 1 week of progressed increasing intensity exercise, M and MR groups were trained on the treadmill with moderate and high intensity for 1 hour every time and 5 times a week which last for 2 weeks and C and R groups didn't do any exercise.R and MR groups were injected with RPM(2mg/kg mass)for 5 times a week and the injection was carried out 1 hour before the beginning of exercise every time. All the rats were killed 48 hours after the last time exercise.Mitochondria were isolated from skeletal muscle,brain and liver which were used for testing ROS production and complexâ… activity.The other part of skeletal muscle were saved in -80℃for testing complexâ… ,â…¡,â…£proteins level and P-Ser-473 Akt,PGC-1αand NRF-2 protein level as well as mtDNA and COXâ…£gene expression.Results:1)There was no significant change of ROS production at rest among C,M and H group in the first 18 rats and among C,R,M and MR groups in the second 24 rats (P＞0.05).2)Complexâ… activity was tested only in the second 24 rats.It increased significantly in M group from mitochondria isolated from all three tissues(brain,liver and skeletal)in the second 24 rats(P＜0.05).The same change also appeared in MR group from live mitochondria.Complexâ… activity decreased significantly in R group from brain mitochondria and there was significant difference between M and R groups(P＜0.05).3)Complexâ… protein expression increased significantly in M and H groups in the first 18 rats.Complexâ…¡protein didn't change among three groups and Complexâ…£protein had the trend to increase in M and H group but without significance.4)Complexâ… protein expression increased in M and MR groups in the second 24 rats.Complexâ…¡had no change among four groups and Complexiv had trend to increase in M and MR groups,but without significance.5)P-Ser473-Akt protein expression increased significantly in M and MR groups and PGC-1αand NRF-2 protein expression increased significantly only in M group.6)mtDNA expression showed trend to increase in M group(P=0.083)in the first 18 rats and in the M and MR groups(P=0.08,0.095)in the second 24 rats.COXâ…£gene expression increased significantly in M group and was significantly different with H group in the first 18 rats and also increased significantly in M and MR groups in the second 24 rats.Conclusions:1)ROS may play an important role in mitochondrial biogenesis. However,there may be difference between the effects of acute and long term exercise on ROS production.Mitochondrial ROS production at rest didn't change either after 2 weeks of exercise or exercise combined with RPM injection.2)Exercise training increased Complexâ… activity in mitochondria from skeletal muscle,brain and liver as well as Complexâ… protein expression in skeletal muscle.RPM inhibited Complexâ… activity in brain tissue but not in skeletal and liver.2 weeks of exercise didn't change Complexâ…¡andâ…£protein level which suggested that longer or different style of exercise might be needed to stimulate their changes.3)Endurance training activated Akt/mTOR signaling by increasing Akt phosphorylation in a RPM independent way.4)Moderate intensity exercise induced mitochondrial biogenesis by stimulating the protein expression of PGC-1αand NRF2.RPM could inhibit the effects and the increase in COXâ…£and mtDNA gene expression induced by exercise to some extent.2 The effects of two kinds of cycling exercise on human skeletal muscle mitochondrial biogenesis and fatty acids oxidation and the related signal gene expression.Method:Nine healthy subjects who didn't exercise regularly for at least 6 months before the test were recruited.Two different styles of cycling exercise(continuous and intermittent)with the same intensity(60%VO_2max)and duration time(90min)were carried out on these 9 subjects on separate day.For continuous exercise(Con),the intensity was kept at constant 60%VO_2maxlevel for 90min and for the intermittent exercise(In),the intensity is consist of 120%VO_2maxfor 12sec and 20%VO_2maxfor 18sec and repeated like this for 90min.The expired gas was collected during exercise which used for analyzing the gas metabolism.Blood and muscle sample were taken before exercise(Pre),immediately after exercise(Post)and 3 hours after exercise(3hs Post) which used for testing fatty acids(FFA),lactate and glucose as well as the gene expression of various transcription factors who closely related to mitochondrial biogenesis and fatty acids can be tested.Results:1)Metabolism indexes,such as RQ,VO₂(L/min),VO₂(ml/min.kg weight), Vent,Vent/VO₂,and HR,RPE etc.were very close to each other in In and Con groups.2)Lactate increased significantly at Post in In and Con(P＜0.05)and returned to base level at 3 hs Post.FFA increased significantly at Post and 3 hs Post in Con and In groups(P＜0.05).Glucose decreased significantly in In and Con at Post(P＜0.01)and 3 hs Post(P＜0.05).3)PGC-1αand PRC mRNA increased after exercise but only show significant 3 hs Post in Con and In Groups and there was significant difference between Post and 3 hs Post.PGC-1αmRNA increased 4.6 and 6.9 folds 3 hs Post in Con and In respectively and PRC increased 3.0 and 3.3 folds.There was no significant change of PGC-1βmRNA.4)NRF-1 mRNA decreased in Con and In groups.NRF-1 mRNA decreased 22%(P＜0.05)and 34%(P＜0.05)at Post and 3 hs Post in Con group and 25%(P＜0.05)and 9%(P＞0.05)in In group respectively.NRF-2 mRNA decreased significantly only in In group which decreased 33%(P＜0.05)and 26%(P＜0.05)at Post and 3 hs Post.It showed trend to decrease but without significance(decreased 14%and 13%at Post and 3 hs Post respectively)in Con group.No significant change happened on Tfam,TFB1M and TFB2M mRNA in Con and In groups.5)AMPKα1 didn't change but AMPKα2 decreased significantly at 3 hs Post in Con and In groups(36%and 43%in Con and In group respectively)and both were different with Post(P＜0.05)。6)PPARαand PPARβmRNA didn't change significantly while PPARγdecreased significantly 3 hs Post in Con(23%)not In group.There is no correlation between the expression of three PPARs subunits.7)PDK4 mRNA showed similar change with PGC-1αand PRC and increased 3.0 and 2.2 folds at Post(P＞0.05)and increased 16 and 6.7 folds 3 hs Post in Con and In groups respectively(P＜0.05).CPTⅠβmRNA decreased at Post but only show significance at 3 hs Post in Con group.LPL mRNA showed trend to decrease at Post and increase at 3 hs Post and both of them had no significance.8)The expression of PGC-1αmRNA was positively correlated with PGC-1βin Con and In groups both at Post and 3 hs Post.The positive correlation also existed between TFB1M and TFB2M mRNA in Con and In groups at 3 hs Post(P＜0.01,＜0.001 respectively)but not at Post.Conclusions:1)Two kinds of cycling exercise(continuous and intermittent)with same intensity(60%VO_2max)and duration time(90min)caused similar stress and similar changes in the gene expression of transcription factors related to mitochondrial biogenesis and fatty acids oxidation.2)The regulations of PGC-1 family members by exercise were different.90 min moderate intensity(60%VO_2max)cycling caused significant increase in PGC-1αand PRC mRNA but no change in PGC-1βmRNA.PDK4 mRNA showed similar change with PGC-1αand PRC.These suggested that acute exercise may promote mitochondrial biogenesis and fatty acids oxidation by activating important transcription factors involved.3)NRF-1,NRF-2,AMPKα2,PPARγand CPT-ⅠβmRNA decreased after 90 min moderate intensity(60%VO_2max)cycling.This suggested that some genes were inhibited after acute exercise or at least were inhibited at some time points after acute exercise.It could not be excluded that they would be activated after short time inhibition. It also suggested that the change after acute exercise might be totally different with that after long term exercise.Summary:1)2 weeks of moderate and high intensity exercise enhanced mitochondrial respiration chain complexes activity and protein expression though had no effect on ROS production at rest.Moderate exercise promoted mitochondrial biogenesis as evidenced by significantly increased Akt,PGC-1αand NRF-2 protein expression, COXâ…£,mtDNA gene expression.2)Two kinds of cycling exercise(continuous and intermittent)with same intensity (60%VO_2max)and duration time(90min)caused similar stress to body and similar changes in FFA,Lactate and Glucose as well as the gene expression of transcription factors related to mitochondrial biogenesis and fatty acids oxidation.3)PGC-1α,PRC and PDK4 mRNA increased significantly after 90 min moderate intensity(60%VO_2mxa)cycling,but PGC-1βmRNA has no change and NRF-1,NRF-2, AMPKα2,PPARγand CPT-ⅠβmRNA decreased significantly.These suggested that mitochondrial biogenesis and fatty acid mobilization and oxidation were induced by a bout of endurance exercise while some other related genes were inhibited or at least inhibited temperally.It could not be excluded that these genes would be activated after short time inhibition.The change after acute exercise might be quite different with that after long term exercise.