Molecular Basis Of Rat Skeletal Muscle Phenotype Adaptation In Response To Endurance Exercise And High-fat Diet

BackgroundSkeletal muscle is composed of slow twitch myofiber and fast twitch myofiber. On the basis of ATPase staining or specific myosin heavy chain(MHC) isoform expression,myofibers are divided into typeâ… (mixed oxidative slow twitch myofibers),typeâ…¡a(glucose oxidative fast twitch myofibers),typeâ…¡d/x(mixed fast twitch myofibers),and typeâ…¡b(glycolytic fast twitch myofibers)fibers.Skeletal muscle not only performs a wide range of movements and plays a central role in the control of whole-body metabolism,but also exhibits remarkable adaptive capability in response to a number of physiological and pathophysiological conditions. Many human metabolic diseases such as diabetes mellitus exhibit muscular atrophy and fiber type transformation.Uncovering the mechanisms of cross-talk between controlling fiber type signaling pathway and controlling fiber size signaling pathway may help us to discover new drugs that target specific mechanism for muscular atrophy/pathological hypertrophy-related diseases,disability rehabilitation and diabetes.Exercise and diet are two important factors to produce skeletal muscle phenotype changes.Our pilot study indicated exercise and high fat-diet affect skeletal muscle fiber composition and size.Calcium ion-activated calcineurin(CaN)-nuclear factor of activated T cells(NFAT) is thought to contribute to the fast-to-slow transitions,whereas AKt/S6K1/GSK-3 contribute to the fiber size.Are the two pathways involved in skeletal muscle phenotype changes induced by exercise and high fat-diet? Is there a link between them? Our aim was to investigate the function role of CaN signaling pathway and AKt signaling pathway as the regulators of muscle fiber phenotype changes under condition of exercise and high fat-diet.Chapter1.Interaction of Endurance Exercise and High-fat Diet On Rat Skeletal Muscle Phenotype【Objective】To determine the adaptive changes of skeletal muscle phenotype in response to short-term endurance exercise and high fat diet in rats so as to study further.【Methods】40 male SD rats(150～180g)were randomly assigned to four groups as follows:control diet sedentary group(Normal group),high fat-diet sedentary group(high fat-diet group),control diet exercise group(Exercise group),high fat-diet exercise group.The control diet(14.5KJ/g) was 13%energy from fat,21%energy from protein and 66%energy from carbohydrate,whereas the high-fat diet((21KJ/g) was a Lard oil-based diet consisting of 59%energy from fat,21%energy from protein, and 20%energy from carbohydrate.The exercised animals performed endurance exercise on a motor-driven rodent treadmill(0 incline and tail electrostimulation) at 28m/min speeds for 6 weeks(6 times a week,one hour each time).After experiment, intraperitoneal glucose tolerance test(IPGTT) was conducted at the sixth week. Spectrophotometry was used to determine fasting blood glucose,plasma triglyceride,plasma free fatty acid,plasma total aminoacid concentration,and radioimmunoassay to measure plasma insulin concentration.Visceral fat weight was calculated by the weight of greater omentum,epididymal fat and perirenal fat. Extensor digitorum longus muscle(EDL) and soleus muscle(SOL) were harvested and weighted.The right muscle applied to morphological analysis by ATPase staining, myosin heavy chain(MHC) isoforms electrophoretic approach and to measurement of succinate dehydrogenase(SDH) activity andβ-Hydroxyacyl-CoA dehydrogenase (HADH) activity by spectrophotometry.【Results】1.6-week high fat diet resulted in an increased visceral fat content and a decreased intraperitoneal glucose tolerance;Endurance exercise obviously decreased high fat diet-induced accumulation of visceral fat(high fat diet group:13.56±6.54g, high fat diet plus exercise group:7.63±2.15g,P＜0.05),whereas it improved high fat diet-impaired intraperitoneal glucose tolerance(high fat diet group:27.14±3.40,high fat diet plus exercise group:21.80±1.67,P＜0.05).2.Endurance exercise increased HADH activity and SDH activity in EDL, whereas high fat diet decreased SDH activity in SOL and increased HADH activity in EDL.3.High fat diet increased fast blood insulin concentration,post-exercise intake of high fat diet did not decrease fast blood insulin concentration(high fat diet group: 15.12±5.91,high fat diet plus exercise group:17.98±7.88 uIU/mL)4.6-week treadmill running elicited fast-to-slow fiber type transformation in the SOL and high fat diet tended to prevent this kind of training-induced fiber transformation in SOL.6-week treadmill running and high fat diet had no effect on fiber composition in EDL.5.Endurance exercise and high fat diet increased SOL mass in growing rats without changes of fiber cross-section area.An interaction of increased EDL mass between high fat diet and endurance exercise was observed.Endurance exercise increased typeâ… fiber cross-section area and high fat diet decreased typeâ… ,typeâ…¡b fiber cross-section area in EDL.6.Fast blood insulin concentration was relative to SOL mass(r_s=0.37,P= 0.040),but not Sol fiber type,EDL mass and EDL fiber type.【Conclusion】1.High fat diet impairs intraperitoneal glucose tolerance,whereas endurance exercise improves it.1.Endurance exercise increases fatty acidβoxidative activity and mitochondrial oxidative activity simultaneously and high fat diet increases fatty acidβoxidative activity only.2.Endurance exercise results in fast-to-slow fiber type transformation in the SOL.3.Endurance exercise and high fat diet affect skeletal muscle mass and muscle fiber cross-section area.Chapter2.The Effects of Endurance Exercise and High-fat Diet on Calcineurin andAkt Signaling Molecular in Rat Skeletal Muscle【Objective】To investigate the relationships between skeletal muscle phenotype change and CaN signaling pathway and AKt signaling pathway under condition of endurance exercise and high fat diet.【Methods】Skeletal muscle specimen were the same as chapter1.The left muscle applied to the protein content measurement of phospho-Akt,phospho-S6K1,GSK-3,CaN, and nuclear NFATc₁ by western blotting and measurement of skeletal muscle CaN activity by Colorimetry and skeletal muscle GSK-3 activity by isotopic method.【Results】 1.There was an interaction of an increased SOL basal phosphorylation of Akt at Ser473[p-Akt(Ser473)]protein content between endurance exercise and high fat diet,and also an increased EDL p-S6k1(Thr389) protein content.Endurance exercise improved high fat diet-impaired SOL p-Akt(Ser473),EDL p-S6k1(Thr389) protein protein content,whereas high fat diet increased SOL p-S6k1(Thr389) protein content.2.Endurance exercise and high fat diet increased EDL cytoplasmic GSK-3 protein content.Endurance exercise decreased SOL nuclear GSK-3 protein content.3.High fat diet increased SOL nuclear GSK-3 activity,Endurance exercise decreased EDL cytoplasmic GSK-3 activity.An interaction of SOL nuclear GSK-3 activity was observed between endurance exercise and high fat diet.4.SOL basal CaN activity was not affected by endurance exercise and high fat diet.Endurance exercise increased EDL cytoplasmic CaN activity,and high fat diet decreased EDL cytoplasmic CaN activity.5.Endurance exercise decreased SOL nuclear NFATc₁ protein content.An interaction of ED nuclear NFATc₁ protein content was observed between endurance exercise and high fat diet.Endurance exercise increased nuclear NFATc₁ protein content in EDL.High fat diet impaired endurance-induced increase in nuclear NFATc₁ protein content in EDL.【Conclusion】1.Endurance exercise increases EDL basal cytoplasmic CaN activity,whereas high fat diet decreases EDL cytoplasmic CaN activity.Endurance exercise and high fat diet don't affect SOL basal CaN activity.2.Endurance exercise and high fat diet affect AKt/S6K1/GSK-3 signaling pathway in a muscle-specific fashion.Chapter3.The Effects of Cyclosporine A on Skeletal Muscle PhenotypeAnd Related Signaling Moleculars in Endurance Exercised Rats 【Objective】To investigate the functional role of CaN as a regulator of skeletal phenotype changes under condition of endurance exercise,and the effect of CaN on AKt signaling pathway【Methods】16 exercised rats(exercise protocol was performed following chapter 1 instruction) were randomized into 2 groups at the end of the 3rd week.Half exercised rats(Cyclosporin A group) were treated with Cyclosporin(CSA, 15mg/Kg.BW/day) by hypodermic administration for 3 weeks,the other exercised rats with saline(exercise group).In addition,Normal group in experiment 1 was used as sedentary control group in this experiment.After experimental period,all specimen harvesting and indicator examination protocol was followed experimet 1 instruction.【Results】1.Cyclosporin A prevented endurance exercise induced fast-to-slow fiber type transformation in the SOL(MHCI ratio in exercise group and exercise plus Cyclosporin A group:93.4±6.0%,79.3±8.4%respectively,P=0.007) and decreased typeâ… fiber cross-section area(normal group,exercise group and exercise plus Cyclosporin A group3763.0±386.9,4047.2±201.7,2753.5±244.3μm² respectively, P=0.000).2.Endurance training with cyclosporin A resulted in an increased myofibril protein content and mass of EDL,but fiber type composition underwent no obvious changes and fiber cross-section area decreased.(Typeâ… in normal group,exercise group and exercise plus Cyclosporin A group:1296.8±331.7,1530.5±133.3,902.8±99.2μm²,P=0.001;typeâ…¡ax:2436.5±420.4,2587.2±294.8,1797.5±174.5μm², P=0.002;Typeâ…¡b:4694.2±238.7,4345.2±372.8,2527.5±165.8μm²,P=0.000)3.Cyclosporin A with endurance training had no effect on basal p-Akt(ser473) protein content in SOL and EDL,and also basal p-S6k1(Thr389) protein content in SOL,but there was an increase in p-S6k1(Thr389) protein content in EDL. Cyclosporin A did not affect GSK-3 protein content and activity in response to exercise.【Conclusion】1.Cyclosporin A prevents endurance exercise induced fast-to-slow fiber type transformation in the SOL and decreases muscle fiber cross-section area.2.Cyclosporin A has no effect on skeletal muscle basal P-Akt(Ser473) protein content and GSK-3 activity in response to exercise.Conclusion1.CaN activity is required for endurance exercise induced-fast-to-slow fiber type transformation.2.CaN is involved in regulation of fiber type cross-section area.On the other hand,cyclosporin A increases EDL myofibril protein content in exercised rats.3.AKt/S6K1/GSK-3 signaling pathways are associated wih skeletal muscle mass and fiber cross-section area in a muscle-specific fashion during endurance exercise and high fat diet.4.CaN has no effects on basal P-Akt(Ser473) protein content and GSK-3 activity in response to exercise in skeletal muscle.