The Effects And Underlying Mechanisms Of Resveratrol On Sarcopenic Obesity

BackgroundSkeletal muscle accounts for almost 40%of the total body mass,and the maintenance of healthy skeletal muscles is vital for human locomotion,preventing metabolic disorders and promoting healthy aging.Sarcopenia is the age-related skeletal muscle disorder involving the progressive loss of muscle mass,strength,and functional capacity,which causes physical functional decline,frailty,falls and poor quality of life in older adults.Accumulating evidence suggests that obesity and saturated fatty acids are independent risk factors for sarcopenia and can exacerbate sarcopenia progression.This coexistence of diminished muscle mass and excessive fat accumulation in older adults,recently defined as sarcopenic obesity,has been reported to increase the risk of metabolic syndrome,cardiovascular disease and mortality than either sarcopenia or obesity alone.With the rapidly aging population and increasing prevalence of obesity worldwide,sarcopenic obesity has become a serious public health concern,which imposes tremendous burdens on aged service authorities and healthcare systems.To date,the pathophysiological mechanisms underlying the development of sarcopenic obesity are not well understood and there are no satisfactory strategies for its management.Hence,further understanding of the pathophysiological process of sarcopenic obesity and development of effective and safe approaches are urgent medical needs to promote healthy aging and extend life expectancy.In the past several years,a growing body of literature has highlighted the important role of mitochondrial dysfunction and oxidative stress in the development and progression of sarcopenia.Moreover,some studies have demonstrated that the ectopic lipid deposition in skeletal muscle is associated with metabolic abnormalities and reduced muscle mass and strength.Excessive accumulation of intracellular lipids and their intermediate metabolites in muscle cells could lead to lipid-toxic environment,resulting in impaired mitochondrial function and enhanced reactive oxygen species production.Mitochondria are important sites for fatty acid oxidation.Mitochondrial dysfunction could result in a reduction of fatty acid oxidation capacity,leading to further intracellular lipid accumulation,thus creating a vicious cycle of lipotoxicity and mitochondrial dysfunction.Therefore,identification of the role of mitochondrial dysfunction,oxidative stress and skeletal muscle lipid deposition in the development of sarcopenic obesity and its molecular regulatory mechanism,as well as the development of strategies that could alleviate mitochondrial dysfunction,oxidative stress and lipid deposition,might have significant implications for the management of sarcopenic obesity.AMP-activated protein kinase（AMPK）,a sensor of the cellular energy status,acts as a key regulator of mitochondrial function and oxidative stress in skeletal muscle.Liver kinase B1（LKB1）,a major upstream AMPK kinase,has been shown to regulate mitochondrial biogenesis and antioxidative pathways in skeletal muscle.LKB1 can be activated through phosphorylation at Ser431 by Protein kinase A（PKA）.Therefore,targeting the PKA/LKB1/AMPK signaling pathway might constitute a plausible approach for enhancing mitochondrial function and blocking oxidative stress in skeletal muscle.However,the role of PKA/LKB1/AMPK signaling pathway in sarcopenic obesity remains unclear.Carnitine palmitoyl transferase-1（CPT-1）is an important rate-limiting enzyme in mitochondrial fatty acid oxidation that transports long-chain fatty acids into mitochondria forβ-oxidation.Overexpression of CPT-1 in skeletal muscle could enhance fatty acid oxidation and reduce intramuscular lipid accumulation.Peroxisome proliferator-activated receptor-delta（PPARδ）,a ligand-activated transcription factor,is a potential therapeutic target for metabolic syndrome and related diseases.PPARδagonist has been shown to increase the expression of its target gene CPT-1 in skeletal muscle cells,which leads to the improvement in fatty acid oxidation and the subsequent inhibition of lipid deposition in skeletal muscle.Hence,PPARδ/CPT-1 signaling pathway might be a pharmacological target for improving ectopic lipid deposition in skeletal muscle.However,no information is available regarding the role of PPARδ/CPT-1 signaling pathway in sarcopenic obesity.Although several potentially interesting therapeutic targets for treating muscle atrophy have already been identified,no specific drugs have been approved for the clinical treatment of sarcopenia.Drugs such as ghrelin,insulin-like growth factor I,clenbuterol and testosterone have been shown to prevent muscle atrophy under several wasting conditions,but their clinical applications are limited because of concerns regarding potential side effects,such as an increased risk of developing water-sodium retention,cardiac arrhythmias,prostate hypertrophy and insulin resistance.Therefore,further research is needed to develop effective and safe therapeutic avenues for sarcopenia.Natural products are secondary metabolites preserved by natural organisms in the evolutionary process,and have been served as a valuable source of therapeutic agents for diseases.Many medicinal plants and natural products are considered as a safe and cost-effective alternative to synthetic drugs.Resveratrol（RSV）,a natural polyphenol present in many plant species,exerts a broad spectrum of pharmacological effects,including antioxidative,anti-inflammatory,anti-atherosclerosis and antitumor properties.In recent years,some studies have indicated that RSV can attenuate glucocorticoids,adriamycin,tumor necrosis factor-alpha and angiotensin-induced protein degradation and muscle wasting.Moreover,RSV has also been shown to prevent muscle atrophy in several pathological conditions,including cachexia,diabetes and chronic kidney disease.However,no information is available regarding the effects of RSV on sarcopenic obesity.Both in vivo and in vitro studies have demonstrated that RSV improves mitochondrial function and oxidative stress via the activation of AMPK in skeletal muscle.Furthermore,RSV has been shown to prevent high fat diet（HFD）-induced hepatic steatosis through activation of the PKA/AMPK signaling pathway.In addition,RSV has been reported to protect endothelial cells and retinal pigment epithelial cells via the activation of PPARδ.These findings led us to hypothesize that RSV might protect against sarcopenic obesity by reversing mitochondrial dysfunction and oxidative stress in skeletal muscle through the PKA/LKB1/AMPK signaling pathway and attenuating ectopic lipid deposition in skeletal muscle via the PPARδ/CPT-1 signaling pathway.To verify this hypothesis,we evaluated the effects of RSV on HFD-induced sarcopenic obesity in aged rats and palmitate acid（PA）-induced muscle atrophy in myotubes.The role of PKA/LKB1/AMPK signaling pathway in RSV-mediated regulation of mitochondrial function and oxidative stress,as well as the role of PPARδ/CPT-1 signaling pathway in RSV-mediated regulation of lipid deposition in skeletal muscle,were also investigated.Methods:Part Ⅰ:Effects of RSV on muscle atrophy in sarcopenic obesity models in vivo and in vitro1.50 old（aged 18 months）male Sprague-Dawley rats were randomly divided into the following five groups:old rats fed the normal chow diet（CD）for 20 weeks（O）,old rats fed the HFD for 20 weeks（HO）,old rats fed the HFD for 10 weeks and then the HFD supplemented with 0.4%RSV for 10 weeks（HO+R10）,old rats fed the HFD supplemented with 0.4%RSV starting from the time of study initiation and throughout the 20-week experimental period（R20+HO）,and old rats fed a normal CD supplemented with 0.4%RSV starting from the time of study initiation and throughout the 20-week experimental period（R20+O）.In addition,10 3-month-old male Sprague-Dawley rats were selected as young control group and fed with the normal CD for 20 weeks.Grip strength was measured with a grip strength meter.The body fat volume was analyzed using a Quantum FX MicroCT Imaging System.At the end of the 20-week study period,rats were sacrificed under anesthesia.Blood was collected and the serum biochemical parameters were measured using an automatic analyzer.Skeletal muscle tissues were weighed and collected for hematoxylin and eosin（H&E）staining.The myofiber cross-sectional area（CSA）was measured using ImageJ software.2.Rat L6 myoblasts were differentiated to form myotubes,and then treated with 0.75mM PA in the presence or absence of 25μM RSV for 24 h.Thereafter,myotubes were harvested for subsequent experiments.Cell viability was measured by the CCK-8 detection kit.Myosin heavy chain（MHC）protein levels were determined by western blot.The myotube diameters were measured using ImageJ software.Part Ⅱ:The effects and underlying mechanisms of RSV on mitochondrial function,oxidative stress and protein metabolism in skeletal muscle in sarcopenic obesity models1.Effects of RSV on mitochondrial function,oxidative stress and protein metabolism in skeletal muscle1.1 Mitochondrial morphology in the gastrocnemius muscles（GAS）of rats was analyzed by transmission electron microscopy.Mitochondrial network in L6 myotubes was visualized by incubating cells with MitoTracker probes.1.2 Quantitative real-time PCR（qPCR）was performed to determine the mitochondrial DNA（mtDNA）copy number in the GAS muscles and L6 myotubes.1.3 Mitochondrial membrane potential（Δψm）was assessed in the GAS and L6 myotubes using the JC-1 probe.Oxygen consumption rate in L6 myotubes was measured by the Extracellular Flux Analyzer.The activities of complexes I,II,and IV were assessed in the GAS and L6 myotubes using the enzyme-linked immunosorbent assay kit.1.4 Total levels of cellular ROS in myotubes were evaluated using the ROS Assay Kit.Mitochondrial ROS（mtROS）content in myotubes was determined by measuring the mitochondrial-derived O₂^·-levels labeled with MitoSOX Red.The antioxidant enzyme activities,total antioxidative capability,malondialdehyde（MDA）content and protein carbonyl levels were assessed in the GAS and L6 myotubes using the corresponding assay kits.1.5 The protein levels of peroxisome proliferator-activated receptor-gamma coactivator-1alpha（PGC-1α）and mitochondrial transcription factor A（TFAM）,key regulators of mitochondrial biogenesis,as well as the protein expression of mitofusin-2（mfn2）and dynamin-related protein 1（drp1）,were detected by western blot.1.6 The protein levels of key factors involved in protein metabolism were determined by western blot.2.The role of PKA/LKB1/AMPK signaling pathway in RSV-mediated regulation of mitochondrial function,oxidative stress and protein metabolism in skeletal muscle2.1 The levels of PKA/LKB1/AMPK pathway-related proteins in the GAS and L6myotubes were measured by western blot.The effect of RSV on the PKA/LKB1/AMPK signaling pathway was analyzed by using siRNA interference technology.2.2 Myotubes were transfected with siRNAs targeting PKA,LKB1 and AMPK to investigate the role of PKA/LKB1/AMPK signaling pathway in RSV-mediated regulation of mitochondrial function,oxidative stress and protein metabolism in skeletal muscle.3.Effects of ameliorating mitochondrial dysfunction and oxidative stress in RSV-mediated improvement in protein metabolism and muscle atrophy3.1 Myotubes were treated with TFAM siRNA,mfn2 siRNA and drp1 siRNA,and mitochondrial function was reflected by measuring respiratory oxygen consumption rate.To elucidate the role of mitochondrial dysfunction in the development and progression of sarcopenic obesity,and to investigate the role of mitochondrial function regulation in RSV-mediated improvement in protein metabolism and muscle atrophy,the myotube diameter and levels of protein metabolism-related factors and MHC were measured.3.2 Myotubes were treated with the ROS scavenger N-acetyl-L-cysteine（NAC）and mitochondria-targeted antioxidant MitoTEMPO.The level of oxidative stress was reflected by mesuring total ROS content.To clarify the role of oxidative stress in the development and progression of sarcopenic obesity,and to explore the effects of alleviating oxidative stress on RSV-mediated improvement in protein metabolism and muscle atrophy,the myotube diameter and levels of protein metabolism-related factors and MHC were determined.Part Ⅲ:The effects and underlying mechanisms of RSV on ectopic fat deposition in skeletal muscle1.The triglyceride（TG）levels were assessed in the GAS and L6 myotubes using the corresponding assay kits.2.The levels of PPARδand CPT-1 in the GAS and L6 myotubes were measured by western blot.Myotubes were treated with PPARδsiRNA and PPARδagonist to investigate the effect of RSV on the PPARδ/CPT-1 signaling pathway.3.To elucidate the role of PPARδ/CPT-1 signaling pathway in RSV-mediated regulation of ectopic fat deposition in skeletal muscle,TG levels were measured after transfecting with PPARδsiRNA and CPT-1 siRNA.4.To explore the role of PPARδ/CPT-1 signaling pathway in RSV-mediated improvement in muscle atrophy,MHC levels and myotube diameter were measured after transfecting siRNAs targeting PPARδand CPT-1.The main results and conclusions:Ⅰ:RSV protected against HFD-induced sarcopenic obesity in aged rats and PA-induced muscle atrophy in L6 myotubes1.HFD feeding significantly increased body fat content,reduced skeletal muscle mass and myofiber CSA,and decreased grip strength in aged rats,indicating the successful establishment of sarcopenic obesity models in aged rats.RSV supplementation prevented muscle loss and myofiber CSA decrease,abolished excessive fat accumulation and improved grip strength in HFD-fed aged rats,suggesting that RSV supplementation effectively protected against HFD-induced sarcopenic obesity.2.PA notably reduced MHC content and myotube diameter in L6 myotubes,indicating the successful establishment of myotube atrophy models.RSV administration inhibited the PA-induced decrease in MHC content and myotube diameter,suggesting that RSV could attenuate PA-induced muscle atrophy in vitro.Ⅱ:RSV improved protein metabolism and prevented HFD-and PA-induced muscle atrophy by reversing mitochondrial dysfunction and oxidative stress in skeletal muscle via the PKA/LKB1/AMPK pathway1.RSV improved mitochondrial function,oxidative stress and protein metabolism in skeletal muscle via the PKA/LKB1/AMPK pathway1.1 RSV administration restored the HFD-and PA-mediated reductions in the ratios of p-PKA/PKA,p-LKB1/LKB1 and p-AMPK/AMPK in skeletal muscle.Moreover,LKB1siRNA inhibited RSV-induced LKB1 activation accompanied by decreased ratio of p-AMPK/AMPK.Furthermore,PKA siRNA abolished RSV-triggered PKA activation accompanied by decreased ratios of p-LKB1/LKB1 and p-AMPK/AMPK.These results suggested that RSV activated the PKA/LKB1/AMPK signaling pathway in skeletal muscle.1.2 RSV stimulated mitochondrial biogenesis,maintained mitochondrial dynamics,improved mitochondrial morphology,rescued the reduced mtDNA content,prevented the collapse in mitochondrial membrane potential,restored mitochondrial respiratory function,enhanced mitochondrial respiratory chain complex activities,increased antioxidant capacity and alleviated oxidative stress in the HFD-induced sarcopenic obesity models and PA-induced myotube atrophy models.However,PKA siRNA,LKB1 siRNA and AMPK siRNA transfection abolished these effects of RSV in vitro.These results suggested that the protective effects of RSV on mitochondrial function and oxidative stress in skeletal muscle were exerted via the PKA/LKB1/AMPK signaling pathway.1.3 The increased protein degradation,as well as the decreased protein synthesis in skeletal muscles of HFD-fed aged rats and PA-treated myotubes,were all reversed by RSV administration.However,PKA siRNA,LKB1 siRNA and AMPK siRNA transfection abrogated these effects of RSV in vitro.Moreover,RSV-mediated inhibition of PA-induced decreases in MHC protein levels and myotube diameter was blocked by PKA siRNA,LKB1siRNA and AMPK siRNA transfection in vitro.These data indicated that RSV improved protein metabolism and attenuated muscle atrophy through the PKA/LKB1/AMPK signaling pathway.2.RSV improved protein metabolism and muscle atrophy by reversing mitochondrial dysfunction and oxidative stress in skeletal muscle2.1 TFAM siRNA and mfn2 siRNA transfection inhibited RSV-induced mitochondrial respiration recovery,and the transfection of these siRNAs also diminished the effects of RSV on myotube diameter and levels of protein metabolism-related factors and MHC.Drp1 siRNA transfection improved the mitochondrial respiration capacity and attenuated PA-induced protein metabolism imbalance and myotube atrophy,and these effects were similar to those obtained with RSV.These data indicated that mitochondrial dysfunction has an important role in the development and progression of sarcopenic obesity,and RSV inhibited protein degradation,increased protein synthesis and prevented muscle atrophy by reversing mitochondrial dysfunction in skeletal muscle.2.2 NAC and MitoTEMPO markedly decreased cellular ROS levels in PA-treated myotubes.Furthermore,NAC and MitoTEMPO mimicked the effects of RSV on the expression of protein metabolism-related factors and myotube atrophy.These results suggested that oxidative stress can promote the development and progression of sarcopenic obesity,and RSV improved protein metabolism and inhibited muscle atrophy by alleviating oxidative stress in skeletal muscle.Ⅲ:RSV attenuated ectopic fat deposition in skeletal muscle and prevented muscle atrophy via the PPARδ/CPT-1 pathway1.RSV administration rescued the decreased PPARδand CPT-1 protein levels in the HFD-induced sarcopenic obesity models and PA-induced myotube atrophy models.Moreover,PPARδsiRNA inhibited RSV-mediated increased CPT-1 protein expression.These results suggested that RSV activated the PPARδ/CPT-1 signaling pathway in skeletal muscle.2.RSV attenuated the increased TG content in the skeletal muscles of HFD-fed aged rats and PA-treated myotubes.However,RSV-mediated reduction in TG levels was blocked by PPARδsiRNA and CPT-1 siRNA.Moreover,PPARδsiRNA and CPT-1 siRNA transfection abolished the inhibitory effects of RSV on PA-induced decrease in MHC content and myotube diameter.These results indicated that RSV attenuated ectopic fat deposition in skeletal muscle and prevented muscle atrophy via the PPARδ/CPT-1 signaling pathway.In summary,we demonstrated that mitochondrial dysfunction,oxidative stress and ectopic fat deposition in skeletal muscle contributed to the development and progression of sarcopenic obesity.Moreover,RSV prevented HFD-induced sarcopenic obesity by reversing mitochondrial dysfunction and oxidative stress through the PKA/LKB1/AMPK pathway and attenuating ectopic lipid deposition in skeletal muscle via PPARδ/CPT-1 pathway.These findings have important theoretical significance in elucidating the pathophysiological mechanisms of sarcopenic obesity,and also provide evidence showing the potential protective effects of RSV on sarcopenic obesity.