| 1 BackgroundBoth too much energy intake and too little physical activity have led to the increasing obese population worldwide,which is a global public health problem.Currently,the methods of obesity intervention are based on reducing energy intake and/or increasing physical activity.Of them,hypoxic training is a gradually accepted type of obesity intervention because of the characteristics of suppressing appetite and reducing weight faster than normal oxygen exercise.The incredible obese intervention effect of hypoxic training is closely related with the enhanced lipid metabolism.Hypoxic training can enhance lipid metabolism by regulating the expression of lipid metabolism related factors through transcription factors,endocrine system,nervous system and micro RNA.Among them,the regulation of lipid metabolisation-related gene expression at the transcriptional level and post-transcriptional level has become therapeutic targets and research focuses due to its regulatory role in multiple tissues of obese individuals.Skeletal muscle is not only a main motor organ,but also an important site for lipid metabolism.Obesity results in glucose and lipid metabolism disorder,skeletal muscle lipid oxidation imbalance,insulin resistance and skeletal muscle dysfunction.Long-term hypoxic training leads to the increased fatty acid utilization in skeletal muscle.Because skeletal muscle is not a fat storage organ,its fatty acids mainly come from the decomposition of extramuscular tissues,such as adipose tissue.Therefore,the increase of fatty acid utilization in skeletal muscle may affect the body’s overall lipid metabolism.However,it is not clear whether the changes of skeletal muscle lipid metabolism during and after hypoxic training can improve the body composition and blood lipid level of obese individuals.Previous studies have shown that hypoxic training can regulate lipid metabolism through mi R-27/PPARγand mi R-122/PPARβ.In this paper,the expression of lipid metabolism related micro RNA（mi R-27 and mi R-122）in skeletal muscle of obese rats will be well controlled.Systematic investigation will be performed to reveal the effects of mi R-27 and mi R-122expression change on body composition,blood lipids and lipid metabolism related genes regulated by mi R-27/PPARγand mi R-122/PPARβpathways,which may provide scientific instructions for weight and fat reduction in hypoxic training.2 Objective（1）To explore the effects of mi R-27 and mi R-122 expression on body composition,blood lipid and the expression of skeletal muscle PPARγ,PPARβand other lipid metabolism related genes in obese rats during hypoxic training;（2）To elucidate the regulatory mechanism of mi R-27/PPARγand mi R-122/PPARβin skeletal muscle lipid metabolism of obese rats during hypoxic training.3 MethodsA total of 150 three-week-old male Sprague-Dawley rats each were purchased from Beijing Vital River Laboratory Animal Technology Co.,Ltd.and housed under standard specific pathogen-free（SPF）conditions in China Institute of Sport Science.After being given a normal diet for one week,the subjects were randomly assigned to two groups.One group were continued to be fed with a normal diet（N,n=20）.The other group were fed with a high-fat diet（HFD,n=130）for 10 weeks,which were used to select the obese rats.Sixty obese rats were randomly divided into six groups with 10 rats in each group for AAV9 delivery and hypoxic exercise.AAV9-Zs Green was delivered to 10obese rats assigned to the placebo control group（CE）,AAV9-Zs Green-mi R-27b to 10obese rats assigned to the mi R-27 overexpression group（OE1）,and AAV9-Zs Green-mi R-27b-3p inhibitor to 10 obese rats assigned to the mi R-27b inhibition group（IE1）,AAV9-Zs Green-mi R-122 to 10 obese rats assigned to the mi R-122 overexpression group（OE2）and AAV9-Zs Green-mi R-122-5p inhibitor to 10obese rats assigned to the mi R-122-5p inhibition group（IE2）.The remaining 10 obese rats that did not receive any injection were assigned to the hypoxic quiet group（H）.The six groups of obese rats were housed in hypoxic conditions with 13.6%O2.The rats in groups CE,OE1,IE1,OE2 and IE2 were given exercise training on a treadmill at a speed of 20 m/min,for 1 h/day,5 days/week,for a total of 4 weeks,and the rats in group H were not given this opportunity to exercise.After the last training,the rats in groups CE,OE1,IE1,OE2 and IE2 were allowed to recover for 24 hours.Then these rats were sampled after fasting for 12hours as well as those of group H.Spontaneous fluorescence of a frozen section of skeletal muscle was conducted to confirm that r AAV9 was successfully delivered into the skeletal muscle.Serum lipid concentration was determined by using commercially available kits.RT-q PCR was used to detect the gene expression of mi R-27,mi R-122,PPARγ,PPARβand other lipid metabolism related factors gene levels in skeletal muscle of the obese rats.Furthermore,western blot was adopted to detect the protein expression of PPARγ,PPARβand other lipid metabolism related factors in obese rat skeletal muscle.4 Results4.1 Effects of mi R-27 expression changes on body composition,blood lipid level,and the expression levels of PPARγand its downstream lipid metabolism related factors were investigated in the skeletal muscle of obese rats after hypoxic training.（1）Body compositionAfter four weeks of hypoxic training,compared with group H,body composition indexes including body weight,BMI,Lee’s index,perirenal fat weight,epididymal fat weight and fat body ratio were decreased in group CE,and there were no significant differences in body composition indexes among the three groups of obese rats with various mi R-27 expression levels（expression control CE group,overexpression OE1group and expression inhibition IE1 group）.（2）Blood lipidsAfter four weeks of hypoxic training,compared with group H,the levels of serum TG,LDL-C and FFA（p<0.01 p<0.01 and p<0.01）were decreased in obese rats of group CE,and the level of serum HDL-C was increased（p<0.01）;compared with group H,the levels of serum TG,LDL-C and FFA（p<0.01,p<0.01 and p<0.05）were decreased in obese rats of group OE1;compared with group CE,mi R-27overexpression significantly increased the levels of serum TG and LDL-C（p<0.05and p<0.01）,while inhibited mi R-27 expression significantly decreased the level of serum TC（p<0.05）,and increased the level of serum HDL-C（p<0.01）.Compared with groups H and OE1,inhibited mi R-27 expression significantly decreased the levels of serum TC,TG,LDL-C and FFA（p<0.01,p<0.01,p<0.05 and p<0.01;p<0.01,p<0.01,p<0.01 and p<0.01）,and increased the level of serum HDL-C（both p<0.01）.（3）The expression levels of mi R-27 regulated downstream lipid metabolism factors were measured in skeletal muscle of obese rats after hypoxic training,which included PPARγand its downstream factors involving in cholesterol metabolism（ABCA1 and CYP7A1）,fatty acid synthesis（ADD1）,fat metabolism（ATGL and LPL）,and fatty acid transport related factors（CD36,H-FABP,A-FABP and FATP）.a.PPARγ:After four weeks of hypoxic training,there were no significant differences in its expression level between groups H and CE;compared with group CE,mi R-27 overexpression significantly decreased its m RNA expression level（p<0.01）,while inhibited mi R-27 expression increased its m RNA（p<0.01）and protein（p<0.05）expression levels.b.Cholesterol metabolism:After four weeks of hypoxic training,compared with group H,the ABCA1 m RNA expression level was increased（p<0.01）,while the CYP7A1 m RNA expression level was decreased（p<0.01）in group CE;compared with group H,mi R-27 overexpression in skeletal muscle decreased CYP7A1 m RNA expression level（p<0.01）,while inhibited mi R-27 expression resulted in the decreased m RNA expression level of ABCA1（p<0.01）,increased m RNA（p<0.01）and protein（p<0.01）expression levels of CYP7A1 compared with group CE;compared with group CE,mi R-27 overexpression in skeletal muscle of hypoxic exercise obese rats did not change ABCA1 expression level,while inhibited mi R-27expression significantly decreased ABCA1 m RNA expression level（p<0.05）,and increased CPY7A1 m RNA（p<0.01）and protein（p<0.01）expression level;compared with group OE1,inhibited mi R-27 expression significantly decreased ABCA1 m RNA expression level（p<0.05）,and increased ABCA1 protein（p<0.05）expression level,CPY7A1 m RNA（p<0.01）and protein（p<0.01）expression levels.c.Fatty acid synthesis:After four weeks of hypoxic training,there were no significant differences in ADD1 expression level between groups H and CE obese rat skeletal muscles;compared with group H,mi R-27 overexpression in skeletal muscle of hypoxic exercise obese rats did not affect ADD1 m RNA expression level,significantly decreased ADD1 protein expression level（p<0.05）,while inhibited mi R-27 expression decreased ADD1 m RNA expression（p<0.01）compared with groups H,CE and OE1（p<0.05,p<0.01 and p<0.01）,and increased ADD1 protein expression of（p<0.01）compared with group OE1.d.Fat metabolism:Compared with group H,the ATGL m RNA expression level was increased（p<0.01）,while the LPL expression level was not changed siginificantly in group CE;compared with group H,mi R-27 overexpression in skeletal muscle of hypoxic exercise obese rats significantly increased the m RNA levels of AGTL and LPL（p<0.01 and p<0.01）,and did not affect their protein levels,while inhibited mi R-27 expression decreased the m RNA level of ATGL（p<0.01）,and increased the protein levels of ATGL and LPL（p<0.05 and p<0.01）compared with group CE;compared with group OE2,inhibited mi R-27 expression decreased the m RNA level of ATGL（p<0.01 and p<0.05）,and increased the protein levels of ATGL and LPL（p<0.01 and p<0.01）.e.Fatty acid transport:Compared with group H,the CD36 m RNA expression level was increased（p<0.05）,the H-FABP expression level was not changed siginificantly,and the A-FABP m RNA expression level was decreased（p<0.01）in group CE;compared with group H,mi R-27 overexpression in skeletal muscle of hypoxic exercise obese rats significantly increased the m RNA（p<0.01）expression level of CD36;compared with group CE,mi R-27 overexpression in skeletal muscle of hypoxic exercise obese rats significantly increased the m RNA expression levels of H-FABP and FATP（both p<0.01）,and decreased A-FABP m RNA expression level（p<0.01）,while inhibited mi R-27 expression increased the m RNA and protein expression levels of CD36（both p<0.01）,and decreased the m RNA levels of A-FABP and FATP（p<0.05 and p<0.01）.4.2 Effects of mi R-122 expression changes on body composition,blood lipid level,the expression levels of PPARβand its downstream lipid metabolism relatedfactors were measured in obese rats after hypoxic training.（1）Body compositionAfter four weeks of hypoxic training,there were no significant differences in body composition indexes,such as body weight,BMI,Lee’s index,perirenal fat weight,epididymal fat weight and fat body ratio among the three groups of obeses rats with different mi R-122 expression levels（expression control CE group,overexpression OE2 group and inhibited expression IE2 group）.（2）Blood lipidscompared with group H,mi R-27 overexpression in skeletal muscle of hypoxic exercise obese rats significantly decreased the levels of serum TG,LDL-C and FFA（p<0.01,p<0.01 and p<0.05）;Compared with group CE,mi R-122 overexpression significantly increased the levels of serum TG and LDL-C（both p<0.01）,while inhibited mi R-122 expression significantly decreased the level of serum TG（p<0.05）,and increased the level of serum HDL-C（p<0.05）.Inhibited mi R-122 expression significantly decreased the levels of serum TC,TG,LDL-C and FFA（all p<0.01;p<0.01,p<0.01,p<0.01 and p<0.05）and increased serum HDL-C level（p<0.01 and p<0.01）compared with groups H and OE2.（3）The expression levels of mi R-122 regulated downstream lipid metabolism factors were measured in skeletal muscle of obese rats after hypoxic training,which included PPARβand its downstream factors involving in cholesterol metabolism（HMGR）,fatty acid synthesis（ACC and FAS）,and fatty acid oxidation（CPT1）.a.PPARβ:Compared with group H,its m RNA expression level has an increasing tendency in group CE（p>0.05）;compared with groups H and CE,mi R-122 overexpression increased its m RNA expression levels（both p<0.01）,while inhibited mi R-122 expression increased its m RNA and protein expression levels（all p<0.01）compared with groups H,CE and OE2.b.Cholesterol metabolism:There were no significant differences in its expression in skeletal muscle of hypoxic exercise obese rats between groups H and CE;both mi R-122 overexpression and inhibited expression in skeletal muscle of hypoxic exercise obese rats did not affect neither HMGR m RNA nor protein expression levels.c.Fatty acid synthesis:Compared with group H,the ACC m RNA expression level was increased（p<0.05）,while the FAS m RNA expression level was decreased（p<0.01）in group CE;compared with group CE,mi R-122 overexpression increased ACC protein and FAS m RNA levels（both p<0.01）,while inhibited mi R-122expression increased ACC m RNA and protein expression levels（p<0.01 and p<0.05）,and both m RNA（p<0.01）and protein（p<0.01）expression levels of FAS compared with group CE;compared with group OE2,inhibited mi R-122 expression increased ACC m RNA level（p<0.01）and FAS protein level（p<0.01）,and decreased ACC protein level（p<0.05）.d.Fatty acid oxidation:There were no significant differences in CPT1expression level between groups H and CE;compared with group CE,mi R-122overexpression did not affect CPT1 expression level,while inhibited mi R-122expression increased CPT1 expression m RNA level（p<0.01）;compared with group OE2,inhibited mi R-122 expression increased CPT1 m RNA level（p<0.01）.5 Conclusion（1）mi R-27/PPARγcontributed to promote cholesterol metabolism and decrease serum TC and LDL-C levels by regulating the expression of CYP7A1 in skeletal muscle of obese rats through hypoxia training;mi R-27/PPARγpromoted fatty acid transport and contributed to increased fatty acid intake and decreased serum TG and FFA levels iby regulating CD36 expression,but it does not affect the body composition of obese rats.（2）mi R-122/PPARβcontributed to promote fatty acid metabolism and reduce serum TG and FFA levels by regulating the expressions of ACC,FAS and CPT1 in skeletal muscle of hypoxic training obese rats,but it does not affect the body composition of obese rats. |
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