Effects Of Nitrogen Monoxide On Iron Metabolism Of Skeletal Muscle During Different Load Endurance Training In Rats

Middle-distance race is a typical periodicity endurance training,and carbohydrate oxidation is the main way to produce energy for exercise.The utilization efficiency of oxygen can affect the production of ATP in skeletal muscle.Hemoglobin(Hb) in the red blood cell can transport oxygen.Myoglobin(Mb) of skeletal muscle can accept and store oxygen transported by Hb.Cytochrome of electron transfer in mitochondrion can affect the ablity of oxidation. Iron is needed for the synthesization of Hb,Mb and Cytochrome.Iron is utilised by the body for oxygen transport and energy production,and therefore iron is essential to athletic performance.Normal iron status in athletes is especially important during exercise.It has been reported that strenuous bouts of prolonged exercise and heavy training were associated with iron deficiency in both animal models and athletes.It is not known whether different load exercise induce iron deficiency.The molecular regulatory mechanism of the changed iron status and iron metabolism in muscle during exercise is not well determined.Some researches reported nitric oxide(NO) may play an important role in iron redistribution in exercise,while the molecular regulatory mechanism of iron metabolism in skeletal muscle was unclear.The present study investigated the effects of NO on iron metabolism in vivo and in vitro in order to illuminate the molecular regulatory mechanism of iron metabolism in exercise.The experiments in vivo:examined the level of iron status,NO,iron storage and the expression of iron transport related proteins in rats' gastrocnemius after 5-week different load treadmill exercise of progressive loading.The experiments in vitro:examined the effects of SNAP(NO donor) on iron release and intake of L6.We hypothesized that NO plays an important role in iron metabolism in gastrocnemius during exercise.Methods:The experiments in vivo:18 male Wistar rats(300±10g) were randomly divided into three groups:control group(CG),moderately exercised group(MG) and strenuously exercised group(SG).The status of blood,serum iron,serum ferrtin,NO content and NOS activity,non-haem iron in different tissue were examined after 5-week different load treadmill exercise of progressive loading.The expression of transferrin receptor 1(TfR1),divalent metal transporter1(DMTl:DMTl(IRE) and DMT1(non-IRE))and ferroportin 1(FPN1) in gastrocnemius of rats were determined by Western Blot.The experiments in vitro:The L6 cells were treated with SNAP(NO donor)(control group(CG) 0 mM and experimental group(EG) 0.5 mmol/L) for 24 h,and examined the effects of NO on iron release and intake by ⁵⁵Fe.The expression of TfR1,DMT1(IRE),DMT1(non-IRE) and FPN1 of L6 cells were determined by Western Blot.Results:(1) Effects of different load endurance training on rats' weight:The body weight of SG was significantly decreased 19.18%compared with CG after 5-week training(P<0.01).There was no significantly change between CG and MG.(2) Effects of different load endurance training on rats' blood status:The red blood cell count,haemoglobin and haematocrit of SG were decread 32.39%,32.05%and 21.30%than that of CG(P<0.01).While the red blood cell(RBC),Hb and hematocrit(Hct) in MG were increased compared with CG and SG(P<0.01).(3) Effects of different load endurance training on rats' serum iron status and non-haem iron distribution in different tissues:The non-haem iron in gastrocnemius of SG and MG were significantly increased compared with CG(P<0.01).Serum iron,serum ferritin of MG were increased than that of CG.On the contrary,the serum iron,serum ferritin,TS and non-haem iron in bone marrow of SG were significantly decreased compared with CG and MG.(4) Effects of different load endurance training on the expression of TfR1,DMT1(IRE),DMT1(non-IRE) and FPN1 of rats' gastrocnemius:Western Blot showed the expression of TfR1 and DMT1(IRE) were increased in MG and SG than that of CG,while the expression of FPN1 in MG and SG was drcreased comared with CG(P<0.05).There was no significant change of DMT1(non-IRE) among three groups.(5) Effects of different load endurance training on the level of NO and the activity of NOS in serum and gastrocnemius of rats:The level of NO and the activity of NOS in serum of SG were significantly increased than that of CG and MG(P<0.01).The level of NO and the activity of NOS in gastrocnemius of SG were significantly increased than that of CG(P<0.01). The level of NO and the activity of NOS in serum of MG were significantly increased than that of CG(P<0.01),but lower than that of SG(P<0.01).There is a linearity corelation between the level of NO and iron on rats' gastrocnemius in physiological range(r=0.81,P<0.01).(6) Effects of NO on iron release and intake of L6 cells:The iron intake was increased of SNAP cells(P<0.01).But there was no significant change of iron release between the two groups.(7) Effects of NO on the expression of iron transport proteins of L6 cells:The expression of TfR1 and DMT1(IRE) were increased in EG cells than that of CG cells,while the expression of FPN1 was drcreased comared with control cells(P<0.05).But there was no significant change of DMT1(non-IRE) of the expression between the two groups.Conclusions:(1)The NO of rats was increased after moderately exercise.The expression of TfR1 and DMT1(IRE) were increased while the expression of FPN1 in gastrocnemius was decreased,in turn the stored iron in gastrocnemius was increased.There was a linearity corelation between the level of NO and iron of rats' gastrocnemius in physiological range.At the same time,the RBC,Hb content,serum iron and transferrin saturation(TS) were also increased than that of CG and MG,which resulted in the elevated O₂ and iron in the skeletal muscle and the increaed oxidation,which was a physioadaptation to exercise.(2) The NO of rats was rapidly increased after strenuous exercise.Though the non-haem iron of gastrocnemius was increased,the iron in bone marrow was significantly decreased.As a result,the RBC,Hb content,serum iron and TS were significantly decreased,which caused sports hypochrosis.The serum ferritin and the stored iron were decreased.Most of the stored iron in bone marrow was removed to skeletal muscle,while the stored iron was not changed in liver,which showed the iron metabolism was disordered after strenuous exercise.The high load exercise was able to destroy the iron homeostasis.(3) The increased NO induced by exercise might lead to the changed expression of iron-related proteins in gastrocnemius,which resulted in elevated iron absorption and a reduction of iron release.Taken together,these data indicated NO played an important role in iron metabolism of skeletal muscle during exercise,and the regulatory process was related to sports load.Our present results provid the experimental base for the arrangement of training,and preventing exercise-induced hypochrosis.