Cancer cachexia (CC) is a highly debilitating syndrome characterized by loss of body and muscle weight affecting most advanced cancer patients. The receptor for advanced glycation end-products (RAGE) is expressed by several cell types and sustains the inflammatory response in acute and chronic diseases. Total ablation of RAGE (Ager-/- mice) translates into restrained CC and increased survival in tumour-bearing mice. RAGE, which is not expressed in adult healthy myofibres, is re-expressed in atrophying myofibres in cancer conditions. However, the specific contribution of muscular RAGE to CC was unknown. Using an HSA/Cre-loxP system, we generated a tamoxifen-inducible conditional AgermKO mouse model in which RAGE is selectively ablated in myofibres. Tamoxifen-treated AgermKO, Agerflox and Ager-/- mice were subcutaneously injected with Lewis lung carcinoma (LLC) cells, and body changes and survival were monitored until 25 dpi, when histological, molecular and proteomic analyses were performed in tumour-bearing and control mice. Muscle samples of pre-cachectic and cachectic pancreatic cancer patients were analysed to validate the results. Compared with LLC-Agerflox mice, LLC-AgermKO mice showed reduced (7.5% [p = 0.004] vs. 15.1% [p < 0.0001]) body weight loss, no significant reduction of hind-limb muscle mass and strength and myofibre cross-sectional areas, increased survival (69.2% vs. 42.9% mice alive at 25 dpi) and restrained muscle and serum pro-inflammatory factors. Mechanistically, AgermKO muscles resist cancer-induced atrophy by maintaining an active Akt-GSK-3β-PGC-1α pathway, and increasing the synthesis of myosin heavy chain (MyHC)-I and -IIa (71.8% [p = 0.008] and 73.9% [p = 0.002] increase, respectively) along with a 76.3% (p = 0.008) increase in hybrid MyHC-I/IIa myofibres. Distinct proteomic signatures characterize muscles of tumour-bearing mice in dependence on RAGE expression, supporting a protective effect of RAGE ablation in muscles. LLC/AgermKO muscles showed increased amounts of several enzymes involved in glycolysis and glucose catabolism, typical of Warburg metabolism. Noteworthy, muscles of pre-cachectic and cachectic cancer patients showed ~3-fold increase (p < 0.05) in RAGE amounts and reduced Akt-GSK-3β-PGC-1α pathway, compared with healthy control subjects. Our data provide evidence that RAGE engagement at myofibre level drives loss of body and muscle weights and inflammation in cancer conditions. RAGE ablation in muscles confers resistance to CC through myofibre remodeling and glycolytic reprogramming. On the clinical side, the overexpression of RAGE is an early event in muscles of cancer patients, suggesting a role for RAGE in the onset of the cachectic syndrome. Thus, the molecular targeting of RAGE might be useful to counteract cachexia and prolong survival in cancer patients.
Ceramides have garnered considerable attention as pro-aging bioactive lipids implicated in both metabolic dysfunction and musculoskeletal decline. Among these, C18:0 and C24:1 ceramides may play a role in the pathophysiology of sarcopenia, a key manifestation of age-related deterioration. However, their specific contributions to muscle degeneration remain poorly defined. C2C12 myoblasts and primary myoblasts were treated with C18:0 or C24:1 ceramides during differentiation to assess myotube formation, migration and intracellular reactive oxygen species (ROS) levels. Three-month-old C57BL/6 mice received daily intraperitoneal injections of C18:0 or C24:1 ceramides for 4 weeks to evaluate muscle morphology and function. In a human cohort of 165 community-dwelling older adults (≥ 65 years), serum ceramide levels were measured via LC-MS/MS and analysed in relation to sarcopenia parameters. Both C18:0 and C24:1 ceramides significantly impaired myogenic differentiation in vitro, as evidenced by reduced myotube number, total myotube area, average area per myotube, nuclei count per myotube and fusion index, through ROS-mediated mechanisms (with up to an 8.6-fold increase in ROS production). Consistently, C18:0 and C24:1 ceramides markedly downregulated key myogenic markers and inhibited ITGB1-FAK-AKT signalling while promoting nuclear activation of FoxO-associated catabolic pathways. These deleterious effects were attenuated by treatment with the antioxidant N-acetylcysteine. In mice, systemic administration of either ceramide resulted in reduced muscle fibre cross-sectional area in the tibialis anterior (by 20.5% and 20.9% for C18:0 and C24:1, respectively) and soleus muscles (by 18.1% and 16.1%), accompanied by decreased grip strength, shorter grid hanging times and reduced latency to fall in the rotarod test. Clinically, in a cohort of 165 older adults (80.6% female; mean age 75.2 ± 5.2 years in controls and 79.7 ± 4.8 years in the sarcopenia group), serum levels of C18:0 and C24:1 ceramides were 27% and 14% higher, respectively, in individuals with sarcopenia compared to controls (p = 0.001 and 0.018). Furthermore, each standard deviation increase in serum C18:0 and C24:1 ceramide levels was associated with a 2.0- and 1.6-fold increased risk of sarcopenia, respectively (p = 0.003 and 0.040). Our findings reveal that circulating C18:0 and C24:1 ceramides are significantly associated with sarcopenia in older adults, while experimental models demonstrate they promote muscle atrophy through oxidative stress-induced impairment of myogenesis and muscle function. These ceramides may serve as minimally invasive biomarkers and potential therapeutic targets for age-related muscle decline. Interventions aimed at modulating ceramide metabolism could offer new avenues for sarcopenia prevention and treatment in aging populations.
Reduced muscle mass and impaired composition have each been independently associated with worse outcomes in patients with cancer. However, emerging evidence suggests that reduced muscle strength-namely, dynapenia-may be particularly important for prognostication, as it is easier to assess in clinical practice compared to muscle mass. Importantly, muscle mass and composition-as assessed with computed tomography images-may not fully capture key physiological changes and/or reflect whole-body alterations, particularly in patients who remain within the normal range. We investigated the predictive power for mortality of low muscle mass and impaired composition, weight loss (WL) and low strength, as well as their combination, in a cohort of patients with cancer. Baseline data on muscle mass and radiodensity (Hounsfield units [HU]) at the L3 level-assessed using computed tomography-along with 6-month unintentional WL (relevant if ≥ 10% of usual body weight) and muscle strength by handgrip were pooled for 477 patients with cancer (59.1% male, mean age 61.2 ± 12.8 years) from studies conducted in Brazil, Canada and Italy. Patients were categorized by sex and body mass index-specific cutoffs for low skeletal muscle mass index, low skeletal muscle radiodensity, WL ≥ 10% and low handgrip strength. Patients were followed for at least 12 months until death or censoring. During a median follow-up of 43 months (IQR: 28-83), 188 patients died. Kaplan-Meier analysis showed no survival differences for low skeletal muscle index or low radiodensity, regardless of handgrip strength. Only WL ≥ 10% consistently identified patients with poorer prognosis, independently of low handgrip strength. Fully adjusted Cox's regression models showed an independent association only with WL (HR = 1.56 [95% CI: 1.12;2.16]; p = 0.008) and low handgrip strength (HR = 2.07 [95% CI: 1.47;2.92]; p < 0.001), as well as an increased risk for all low handgrip strength/%WL categories. Mortality risk increased across all low handgrip strength/%WL categories. Among the eight risk groups combining low skeletal muscle mass index, WL ≥ 10% and low handgrip strength, only those including WL ≥ 10% and low handgrip strength were significantly associated with higher mortality. Low skeletal muscle mass index contributed to a worse prognosis only when combined with both WL ≥ 10% and low handgrip strength. Similar results were observed when skeletal muscle radiodensity was used in replacement of skeletal muscle mass index. In patients with cancer, muscle strength and WL were stronger survival predictors than muscle mass and composition, reinforcing their relevancy as easily assessed key markers of muscle health.
Patients with end-stage liver disease (ESLD) often present with sarcopenia, defined as loss of skeletal muscle mass and quality, which is associated with reduced quality of life and increased mortality. However, the molecular mechanisms driving sarcopenia in ESLD are not fully understood and there are currently no therapeutic interventions. This study aimed to identify potential circulating factors contributing to sarcopenia progression in ESLD by assessing their role in driving transcriptomic alterations in skeletal muscle. Quadriceps muscle tissue, plasma and serum were obtained from ESLD patients (n = 24) and age/sex-matched healthy controls (HC; n = 18) (Clinical Trial ID: NCT04734496, Ethical Approval 18/WM/0167). Total RNA from snap-frozen vastus lateralis muscle biopsies underwent RNA sequencing (Illumina). Serum concentrations of 60 cytokines were profiled by Luminex and ELISA, with comparisons made both between ESLD and HC, and across ESLD aetiologies (alcohol-related, NAFLD, viral hepatitis, other). In vitro, primary human myotubes (from non-ESLD aged donors, NRES #16/SS/0172) were treated with 10% ESLD or HC plasma (24 h, n = 6 per group) followed by RNA sequencing (BGI Genomics). Differentially expressed genes (p < 0.05, fold-change > 1.5) were identified via Qlucore and DESeq2, and pathway analysis performed using Ingenuity (Qiagen). The impact of physiological concentrations of candidate cytokines (IL-1α, GDF-15 and HGF) on myotube thickness, differentiation and mitochondrial function was assessed by immunofluorescence microscopy, RT-qPCR and metabolic flux assays. In ESLD muscle, 387 and 225 genes were significantly up- and downregulated compared to HC, respectively, with cellular senescence identified as a top dysregulated function. Upstream regulator analysis predicted activation of hepatocyte growth factor (HGF) and interleukin-1 signalling. Subgroup analysis revealed distinct transcriptomic profiles based on disease aetiology. Serum profiling identified 15 cytokines significantly elevated (p < 0.05) and five reduced (p < 0.05) in ESLD, including increased HGF and reduced interleukin-1 receptor antagonist. Stratified analysis also revealed aetiology specific cytokine profiles, with only GDF-15 significantly (p < 0.0001) elevated in all groupsTwenty-four-hour ESLD plasma treatment induced 423 differentially expressed genes in human myotubes, which were again associated with significant activation of senescence pathways, with IL-1 identified as a key upstream driver. In vitro, IL-1α, GDF-15, and HGF significantly reduced myotube thickness, nuclear fusion index and perturbed metabolism (increased glycolysis, impaired oxidative phosphorylation). Collectively, these findings suggest that sarcopenia in ESLD is driven by aetiology-specific mechanisms, highlighting the potential for targeted therapies to improve muscle mass and function.
High-intensity interval training (HIIT) with or without L-citrulline (CIT) oral supplementation improves body composition, functional capacities and muscle health in obese older adults, potentially through microRNA-driven regulation. We aimed to (1) investigate the impact of a 12-week HIIT on the expression level of microRNAs in vastus lateralis muscle biopsies and serum of obese older adults and (2) assess whether the differential expression level of microRNAs was associated with clinico-biological adaptations to HIIT and provide potential biomarkers of HIIT response. In this secondary exploratory analysis of a double-blind randomized trial, 36 women and 32 men (67.2 ± 5.2 years) following 12 weeks HIIT randomized in two groups were supplemented daily with CIT (HIIT-CIT, n = 37) or with placebo (HIIT-PLA, n = 31). Phenotypic variables, serum parameters, muscle biopsies and subcutaneous abdominal adipose tissue outcomes were collected pre-intervention and postintervention. To assess the microRNA profile, the miRNome of muscle biopsy and serum was analysed using next generation sequencing in participants' subsets (n = 13). The microRNAs' differential expression level was analysed pre-intervention and postintervention by TaqMan-real-time qPCR in 68 participants. The expression of myo-microRNAs (miR-133a, b, -1, -206) and muscle-related-microRNAs (miR-499, -208) was not altered following HIIT with or without CIT. In HIIT-PLA, HIIT-CIT and subgroups (based on sex, age, body mass index, dynapenic status), the change in muscle (miR-504-5p, -744-5p, -151a-3p, -106b-5p, -127-5p) and circulating (miR-4433b-5p, 151a-3p, -744-5p, 483-3p, -106b-5p, -484) microRNA levels was associated with changes of clinico-biological parameters. Supplementing HIIT with CIT decreased the muscle miR-504-5p level (p = 0.022), correlating with lower body fat, improved functional capacities, muscle power and increased IGF-1 level (r = -0.6, p < 0.05). MiR-744-5p expression increased in dynapenic participants (p = 0.04), associated with lean mass gain (r = 0.50, p < 0.05), while miR-151a-3p downregulation in men's muscle (p = 0.01) was associated with better insulin sensitivity (HOMA-IR, r = 0.57, p = 0.05). In serum, miR-151a-3p upregulation in women (p = 0.01) correlated with improved muscle power and lower circulating leptin levels, while miR-4433b-5p downregulation (p = 0.001) was linked to reduced fat mass, lean mass gain and enhanced functional capacity. The downregulation of miR-106b-5p (p = 0.05) was associated with a higher adiponectin level and a better score of the 4-m walking test (p = 0.05). HIIT did not impact the expression level of myo-microRNAs and muscle-related microRNAs but induced changes in muscle-nonspecific microRNAs in muscle biopsy and serum. Modulations of microRNAs in muscle (miR-504-5p, -744-5p) and serum (miR-151a-3p, -4433b-5p, -106b-5p) were associated with HIIT's beneficial effects, suggesting their role in the HIIT effects and their potential as candidate biomarkers for response.
Sarcopenia is a progressive and systemic skeletal muscle disorder characterized by the decline of muscle mass and function. Despite its early-stage, 'possible sarcopenia' has been emphasized for prompt intervention; there are currently no specific biomarkers for the diagnosis and treatment. RNA sequencing of human skeletal muscle across sarcopenia stages identified CCN5. Adeno-associated virus (AAV) is intramuscularly injected into young C57BL/6J mice to knockdown CCN5 in skeletal muscle. Phenotypic alterations are assessed through behavioural testing, body composition analysis, oil red O staining and transmission electron microscopy. Mechanisms were investigated in C2C12 myotubes using lentiviral infection, Western blotting, immunofluorescence, cellular electron microscopy and seahorse assays. Finally, aged C57BL/6J mice received intramuscular AAV injections to overexpress CCN5 in skeletal muscle, evaluating its therapeutic efficacy against sarcopenia. Clinical samples included 56 participants (48.2% female; mean age: 63.21 ± 8.76 years). By comparing gene expression in human skeletal muscle across three stages of sarcopenia, we identified CCN5 as a gene exhibiting decreased protein expression in possible sarcopenia stage (approximately 33% reduction, p = 0.0245), with this decline persisting into sarcopenia stage (p = 0.0093). In young mice, CCN5 knockdown induced a sarcopenia-like phenotype, encompassing skeletal muscle dysfunction and myosteatosis (fat mass: p < 0.01; intramyocellular triglyceride: 66.02 ± 3.798 vs. 104.5 ± 8.542 μg/mg tissue, p < 0.01). CCN5 deficiency also impaired mitochondrial content and function, particularly by reducing lipid droplet-mitochondrial (LD-Mt) interaction (approximately 47% reduction, p < 0.05). In C2C12 cells, CCN5 knockdown disrupted lipid metabolism (particularly reduced lipid oxidation, CPT1A: approximately 56% reduction, p < 0.001), promoted lipid accumulation and compromised mitochondrial content and function. Mechanistically, secreted CCN5 enhanced LD-Mt interaction and stimulated mitochondrial biogenesis by activating nuclear β-catenin translocation to enhance FOXO3A-dependent transcription, while intracellular CCN5 mitigated myosteatosis by inhibiting PPARγ signalling. In aged mice, CCN5 overexpression improved skeletal muscle function, reduced myosteatosis (fat mass: p < 0.001; intramyocellular triglyceride: 175.0 ± 11.18 vs. 92.18 ± 10.53 μg/mg tissue, p < 0.001) and restored mitochondrial function. CCN5 mitigates myosteatosis and counteracts sarcopenia by promoting mitochondrial biogenesis and enhancing LD-Mt interactions through dual pathways, positioning it as a promising therapeutic target for muscle aging and sarcopenia.
Cachexia and sarcopenia are prevalent, inflammation-linked syndromes in chronic liver disease that worsen outcomes. To our knowledge, their coexistence in a single chronic liver disease cohort has not been systematically examined. In this study, we evaluated the prevalence, clinical features and prognostic impact of cachexia and sarcopenia-alone and combined-in chronic liver disease. We retrospectively screened 776 patients with liver cirrhosis (LC) and/or hepatocellular carcinoma (HCC) at Hokkaido University Hospital (August 2014-May 2025). The inclusion criteria were grip strength, CT-based muscle mass and complete clinical data, yielding 307 patients; 469 did not meet one of the inclusion criteria. Cachexia was determined following the Asian Working Group for Cachexia criteria, and sarcopenia was determined following Japan Society of Hepatology guidelines. Patients were grouped as no cachexia/sarcopenia, cachexia only, sarcopenia only or cachexia+sarcopenia. The outcomes were overall survival, time to liver-related events and time to readmission (Kaplan-Meier and Cox-proportional models). Among 776 patients, 307 were included in the final-analysis. Of 307 patients, 206 (67.1%) were male, the median age was 70 years (range, 19-90 years), 262 patients (85.3%) had LC and 188 patients (61.2%) had HCC. The patients were grouped as no cachexia/sarcopenia (213; 69.4%), cachexia only (54; 17.6%), sarcopenia only (17; 5.5%) and cachexia+sarcopenia (23; 7.5%). The combined group compared with the others had the lowest body mass index, psoas-muscle-index and grip strength (all p < 0.001). Overall survival (OS), liver-related events, LC progression and readmissions were compared between 246 patients with and without cachexia or sarcopenia, after excluding those who visited the hospital on or after July 2023 and had ≤ 3 months of follow-up. OS was shorter in the cachexia only (median 61.8 [95% CI 40.90-not reached (NR)] months, p = 0.046) and cachexia+sarcopenia (median 59.6 [95% CI 14.26-NR] months, p = 0.027) groups than in the no cachexia/sarcopenia group. Multivariable analysis showed that cachexia+sarcopenia (hazard ratio 2.48, p = 0.010), HCC (hazard ratio 3.40, p < 0.001) and diabetes mellitus (hazard ratio 1.80, p = 0.013) independently predicted mortality. The combined group compared with the other groups had a shorter time to liver-related events and readmission. The coexistence of cachexia and sarcopenia-rather than either alone-can be used as an indicator for identifying patients with chronic liver disease at the highest risk of poor outcomes. Concurrent assessment and early, targeted interventions may improve outcomes in this population.
Respiratory fitness and sarcopenia status have been reported to be cross-sectionally linked with each other and increase mortality risk. However, little is currently known regarding the connection between impaired respiratory function and sarcopenia and their joint effect on future death risk. This study included 12 027 participants (50.8% were women; mean [SD] age, 58.9 [9.3] years) from a nationwide, prospective cohort in China (China Health and Retirement Longitudinal Study). Respiratory function was assessed by peak expiratory flow (PEF). Sarcopenia status was assessed according to the Asian Working Group for Sarcopenia 2019 (AWGS 2019) criteria. Time-to-event survival analyses and causal mediation analyses were conducted to assess the joint association of sarcopenia and reduced PEF with all-cause mortality. During a mean follow-up of 8.64 years, 1536 deaths were recorded. After multivariable adjustment, the hazard ratios (HRs) for mortality were 1.51 (95% CI: 1.33-1.71) for possible sarcopenia and 1.67 (95% CI: 1.43-1.94) for diagnosed sarcopenia. A dose-dependent association between PEF and mortality was observed (p-nonlinearity > 0.05). The adjusted HRs per 1-SD decrease in PEF were 1.29 (95% CI: 1.18-1.41), 1.15 (95% CI: 1.03-1.29), and 1.49 (95% CI: 1.30-1.71) among individuals with nonsarcopenia, possible sarcopenia, and diagnosed sarcopenia, respectively (p interaction = 0.127). Causal mediation analysis demonstrated a bidirectional mediation effect, with both natural direct and indirect effects being statistically significant. Diagnosed sarcopenia was associated with excess mortality partly mediated by reduced PEF (total effect HR: 1.63, 95% CI: 1.38-1.92; natural indirect effect HR: 1.10, 95% CI: 1.07-1.14; proportion mediated: 18.5%). Conversely, sarcopenia mediated 10.0% and 7.0% of the reduced PEF-mortality pathway for possible and diagnosed sarcopenia, respectively. Compared with participants who had normal PEF and no sarcopenia, the adjusted HRs (95% CIs) for mortality were 1.52 (1.22-1.89) for possible sarcopenia with normal PEF, 1.73 (1.28-2.35) for diagnosed sarcopenia with normal PEF, 1.60 (1.36-1.88) for impaired PEF without sarcopenia, 2.22 (1.87-2.64) for impaired PEF with possible sarcopenia, and 2.44 (2.01-2.97) for impaired PEF with diagnosed sarcopenia. No significant risk heterogeneity was observed across sex, age or lifestyle subgroups. Sarcopenia and impaired respiratory function are interrelated and jointly elevate mortality risk in middle-aged and older Chinese adults.
Muscle atrophy, characterized by progressive loss of muscle mass and function, is driven by muscle-specific E3 ligases MAFbx and MuRF1. While transcriptional regulation of E3 ligases is documented, the mechanism of their regulation by the ubiquitin-proteasome system remains unclear. This study aims to identify a deubiquitinase (DUB) regulating these E3 ligases and reveal the mechanisms underlying the mitigation of muscle atrophy through inhibition of the discovered DUB. Differentiated C2C12 myotubes were screened using siRNAs to identify DUB genes that can regulate muscle atrophy. Muscle fibre cross-sectional area (CSA), grip strength and gene expression (MAFbx, MyoD, etc.) were evaluated in muscle atrophy-induced mouse model. Human translational relevance was analysed using GTEx skeletal muscle data. We identified that OTU DUBs family genes are increased (log2 FC > 1, p < 0.05) in DEX-induced muscle atrophy. Pharmacological (ubiquitin isopeptidase inhibitor I, G5) and genetic inhibition of YOD1 alleviated DEX- and denervation-induced muscle atrophy by MAFbx destabilization. The UBX domain of YOD1 was found to interact with the LZ domain of MAFbx, and YOD1 stabilized the MAFbx protein by removing polyubiquitin chains at lysine 48 in MAFbx. In in vivo mouse models, G5 treatment effectively ameliorated DEX- or NTX-induced muscle atrophy. Specifically, G5 increased grip strength by 37.64% (DEX, p < 0.0001) and 36.37% (NTX, p < 0.01), while muscle fibre size was improved by 35.85% (DEX, p < 0.01) and 30.76% (NTX, p < 0.0001). These improvements were accompanied by the restoration of MyoD and eIF3-f expression. Consistently, GTEx-based analysis revealed that high YOD1 expression in human skeletal muscles is significantly associated with an increased proportion of smaller fibres (< 2000 μm2), correlating with enriched proteostasis (NES = 1.51)-related and muscle development (NES = -1.44)-related transcriptional signatures. Our study indicates that YOD1 inhibition destabilizes MAFbx protein levels, leading to protection against DEX- and denervation-induced muscle atrophy. Integration of human GTEx data further supports the translational relevance of YOD1 as a regulator of muscle fibre homeostasis. This study provides new insights into the post-translational regulation of muscle-specific E3 ligases and presents evidence showing that targeting YOD1 is a promising therapeutic approach for the prevention and treatment of muscle atrophy.
Sarcopenia has emerged as a potential prognostic factor in patients with advanced prostate cancer (PCa), requiring interventions for its prevention and treatment. We aimed to systematically identify, critically assess and synthesize the available evidence on the effectiveness and safety of interventions for preventing or treating sarcopenia in advanced PCa patients. MEDLINE, Embase and Web of Science were searched. Randomized and non-randomized controlled trials or longitudinal observational studies with a control group focusing on PCa patients aged 60 years and older were considered. Study selection, data extraction and risk-of-bias assessment of the included studies were performed in duplicate. When possible, pooled effect estimates were calculated. Twenty studies (n = 1275) were included. Resistance training (RT) (MD = 3.22 kg; 95% CI 0.69, 5.75) and the use of antimyostatin peptibody (MD = 2.2 kg; SE 0.8%) demonstrated statistically significant prevention of lean body mass loss in men undergoing androgen deprivation therapy (ADT). Exercise improved leg press (MD = 25.17 kg; 95% CI [8.71, 41.62]), leg extension (MD = 9.63 kg; 95% CI [4.83, 14.42]), seated row (MD = 4.38 kg; 95% CI [1.54, 7.22]) and chest press strength (MD = 1.70 kg; 95% CI [-1.48, 4.88]) and enhanced patients' physical functioning in chair sit-to-stand tests (MD = -1.02 kg; 95% CI [-1.70, -0.34]). RT improved health-related quality of life (HRQoL) in both general and specific domains and also reduced somatization (MD = -0.69 kg; 95% CI [-1.32, -0.07]) and psychological distress (MD = -1.63 kg; 95% CI [-3.10, -0.15]). The findings highlight the potential benefits of RT and selected pharmacological interventions on muscle-related and functional outcomes. However, the significant heterogeneity and lack of comprehensive outcome reporting underscore the need for more standardized and long-term research through larger, well-designed randomized controlled trials with standardized measurement methods to draw conclusive evidence and enhance the reliability and applicability of findings in clinical practice.
Skeletal muscle is a postmitotic tissue dependent on a complex and tightly regulated regeneration process involving numerous intracellular and extracellular factors, including myogenic regulatory factors (MRFs), cytokines and myokines. Quiescent satellite cells are activated by physiological stimuli, injury or other traumatic insults for the repair of injuries or growth of the tissue. Activation of satellite cells induces proliferation and expression of MRFs, which in turn activate myogenic differentiation transcription programmes. Transitioning into and committing to terminal differentiation are regulated by myogenin and cell cycle exit markers, notably Rb1 and p21. Differentiation is then complete with the formation of new muscle fibres which incorporate into existing fibres. Upon ageing, the efficiency of differentiation is reduced as a consequence of a loss in the physiological balance between pathways regulating satellite cell quiescence and activation, notably the Notch and Wnt pathways, and increased senescence of the satellite cell pool. Extracellular factors involved in the dysregulation of differentiation upon ageing include low-grade chronic inflammation and remodelling of the extracellular matrix by fibro-adipogenic progenitor cells, thereby negatively affecting the differentiation capacity of satellite cells, resulting in either premature differentiation or senescence. These ageing-associated alterations in muscle homeostasis appear to be amplified in inclusion body myositis (IBM), an idiopathic inflammatory myopathy that almost exclusively manifests in individuals over 45 years of age, making it a prototypical age-related muscle disease. IBM is characterised by chronic inflammation, progressive muscle degeneration and premature ageing of both muscle tissue and the satellite cell niche. Studied with immunohistochemical techniques and multi-omics, muscle biopsy tissue demonstrated increased expression of MRFs as well as increased expression of senescence and genomic stress markers. IBM primary myoblasts demonstrated premature ageing and senescence and increased activity of the Wnt pathway, though differentiation into multinucleated myotubes did not show notable aberrations in signalling pathways or differentiation efficiency. In conclusion, ageing and chronic inflammation lead to dysregulation of key pathways that, in turn, alter the capacity of satellite cells to activate and proliferate, leading to prematurely aged satellite cells that still retain their capacity to differentiate into myofibres. Though in IBM there is an increased abundance of active differentiation markers, reflecting a regenerative response to the massive, sustained muscle atrophy, senescence of the satellite cell niche may impair effective regeneration of the lost muscle tissue.
Muscle-adipose aging reflects interactions between body composition changes and chronic inflammatory processes, but age-related patterns in these measures across ethnically diverse populations remain incompletely described. Understanding population-specific patterns is crucial for developing tailored prevention and intervention strategies. We aimed to characterize age- and sex-specific patterns of muscle-related measures, adiposity, inflammatory markers and lipolysis-related biomarkers within large cohorts from the United Kingdom, the United States and Taiwan, and to examine how these observed patterns varied across cohorts. In this cross-cohort comparative study, we analysed data from UK Biobank (n = 35 436), US NHANES (n = 9157) and Taiwan NAHSIT (n = 4959). We examined age-related trajectories of muscle mass, adiposity, muscle-to-fat ratio, and biomarkers of inflammation and lipolysis. Inflammaging was assessed using C-reactive protein (CRP) and insulin-like growth factor-1 (IGF-1) decline profiles. Lipolytic activity was evaluated using circulating ketone bodies (β-hydroxybutyrate, acetoacetate, acetone) in the UK cohort. Age-specific patterns were described across populations, with particular attention to masking effects of body mass index (BMI) adjustment and late-life metabolic selection. Muscle-adipose aging patterns differed substantially across populations. Taiwanese men, despite having lower absolute appendicular lean mass, showed steeper cross-sectional differences while maintaining higher muscle-to-fat ratios than other cohorts. Inflammaging patterns were consistent across cohorts, with CRP increasing by up to 90% and IGF-1 declining by 20.3%-23.8% from ages 45 to 79, consistent with a potential link between chronic inflammation and anabolic resistance. BMI-adjusted muscle mass showed greater deterioration with age compared with height-adjusted measures, suggesting conventional assessments may mask sarcopenic progression. Adiposity trajectories peaked earlier in Western populations (55-59 years) than in Taiwanese adults (60-64 years), followed by late-life declines of 10.3%-23.5%, indicative of differential lipolytic activation. In the UK cohort, ketone body concentrations increased with age, suggesting higher fat oxidation in older age groups. Among adults aged 80 years or older, reversals in the muscle-to-fat ratio suggested metabolic selection effects across all populations. Muscle-adipose aging reflects population-specific patterns of inflammatory and lipolytic reprogramming, with metabolic efficiency-rather than absolute tissue quantity-emerging as a key determinant of healthy aging. Establishing ethnically tailored reference standards may improve the precision of aging-related risk stratification and interventions across diverse populations.
Ageing is an inevitable biological process that contributes to increased prevalence of age-associated diseases, including sarcopenia, defined by progressive loss of muscle mass, functional decline and a heightened risk of injury. Developing effective interventions remains a critical clinical priority. This study employed a natural ageing mouse model to investigate whether noninvasive low-intensity pulsed ultrasound (LIPUS), a therapeutic ultrasound, delivered to the abdomen, could alleviate age-related muscle deterioration and whether its effects were linked to gut microbiota modulation. C57BL/6 mice were maintained until 92 weeks of age, after which abdominal LIPUS stimulation was administered for 8 weeks. At 100 weeks, both forelimb and hind limb grip strength were assessed prior to euthanasia. Faecal samples from the distal colon were collected for microbiota profiling, and gastrocnemius muscles were harvested for downstream analyses. Naturally aged mice exhibited sarcopenia-like characteristics, including impaired muscle performance, reduced myofiber diameter and decreased muscle weight (n = 6, p < 0.01, p < 0.001). Age-related renal impairment promoted the accumulation of advanced glycation end products (AGEs) in skeletal muscle, triggering pro-inflammatory signalling cascades characterized by elevated COX-2, phosphorylated NF-κB, NLRP3, IL-1β and Caspase-1 (n = 5-6, p < 0.01). LIPUS treatment significantly improved muscle strength (forelimb and hind limb grip strength, n = 6, p < 0.001, p < 0.01) and muscle mass (n = 6, p < 0.01), while suppressing inflammatory mediators (n = 5-6, p < 0.05). Gut microbiota analysis showed that LIPUS increased microbial diversity (n = 5-6, p < 0.05) and altered taxonomic composition, enriching anti-inflammatory taxa such as Lactobacillus, Bifidobacterium, Faecalibaculum and Coriobacteriaceae_UCG_002 (n = 6, p < 0.05). Correlation analysis indicated that these LIPUS-enriched taxa were positively associated with enhanced muscle performance. These data suggest that LIPUS mitigates sarcopenia in naturally aged mice by restoring muscle integrity and attenuating inflammation, possibly via gut microbiota regulation. This study shows that natural ageing in mice induces sarcopenia-like features with inflammatory activation and gut microbiota alterations. Abdominal LIPUS treatment alleviated muscle loss, reduced inflammation and promoted beneficial microbes, rejuvenating the ageing muscle. These findings highlight LIPUS as a safe, noninvasive and potentially translatable strategy for sarcopenia, warranting further investigation of its microbiota-muscle interactions.
Despite ongoing interest in anabolic therapies for sarcopenia and cachexia, pharmacological interventions are being developed to counteract age- and condition-related losses of muscle mass and strength. This systematic review and meta-analysis evaluated the effect of nandrolone decanoate on lean soft tissue (LST), fat mass, handgrip strength, knee extension strength and bone mineral density (BMD) in adults. Following PRISMA guidelines, we searched PubMed, Scopus, Web of Science and Cochrane Library (inception to April 2025) for randomised controlled trials (RCTs) comparing nandrolone decanoate to placebo in adults ≥ 18 years. Data were analysed using a random-effects meta-analysis, and risk of bias was assessed using the RoB2 tool. Mean differences (MD) alongside 95% confidence intervals (95% CI) were applied, whereas standardised MD (SMD) was used when methods or/and units of measurement were not identical (i.e., body fat mass/percentage). Twenty RCTs were included in this study. Nandrolone decanoate significantly increased LST (k = 11; MD: 1.59 kg, 95% CI 1.06-2.13, p < 0.01, I2 = 0%) but had no effect on fat mass (k = 11; SMD: -0.04, p = 0.65), handgrip strength (k = 12; SMD: 0.39, p = 0.10) or knee extension strength (k = 1; p = 0.99). BMD outcomes were inconsistent, with only total proximal femur BMD showing improvement (p < 0.05). No publication bias was detected. Most studies showed a low risk of bias, whereas GRADE assessment displayed a low certainty of evidence. Although nandrolone decanoate modestly increases LST, this was not accompanied by improvements in handgrip strength or consistently observed improvements in BMD, and functional outcomes were informed by limited trial data. Taken together, these findings do not support routine clinical use of nandrolone decanoate for improving musculoskeletal health. Given its limited benefit and known safety concerns, non-pharmacological approaches such as resistance training and nutritional support should remain the preferred strategy for preserving musculoskeletal health.
Early prevention of sarcopenia through exercise is vital to health, independence and mortality risk in older adults; however, there is a scarcity of evidence concerning the long-term effects of aerobic exercise alone. We tested the primary hypothesis that a 5-year decline in key defining components of sarcopenia is less in an aerobic exercise group compared with a control group. Secondly, we tested the same hypothesis using a model of 'clinically defined sarcopenia'. Norwegian community dwelling older adults (n = 1567; aged 70-75 years at inclusion) participated in the Generation 100 Study. They were randomized in a 1:1:2 ratio-stratified by sex and cohabitation status to either moderate intensity continuous training (MICT, n = 387) or high intensity interval training (HIIT, n = 400), or to a control group following the Norwegian physical activity guidelines (CON, n = 780). Key defining components of sarcopenia were grip strength (kilograms), skeletal muscle index (kg/m2) and gait speed (m/s). Clinically defined sarcopenia was based on the European Working Group on Sarcopenia in Older People and recent normative data for the Norwegian population. An intention to treat linear mixed model and an ordinal logistic regression mixed model were used to examine the effects of aerobic exercise on sarcopenia on the 1, 3 and 5 years follow-up. p values for the main hypothesis were adjusted using the Benjamini-Hochberg correction. Mean age across groups was 72 years of age, including ~50% women, ~50% with higher education, 86%-90% with good or very good health and 73%-76% considered physically active. Compared with CON, grip strength declined less for HIIT at Year 1 (estimated difference of 0.98 kg; p < 0.001) and Year 3 (estimated difference of 0.03 kg; p = 0.016). At Year 3, HIIT increased their gait speed by 0.01 m/s (estimated difference between HIIT vs. CON of 0.03 m/s; p = 0.016). At Year 5, all groups declined from baseline (CON -0.07; MICT -0.10 and HIIT -0.04 m/s). Participants determined as having 'clinically defined sarcopenia' at baseline, Years 3 and 5 were: 22%, 58% and 63% (CON); 20%, 55% and 67% (MICT) and 19%, 50% and 56% (HIIT). Odds for developing clinically defined sarcopenia were significantly lower for HIIT (OR = 0.51; p = 0.018 at Year 3; OR = 0.47; p = 0.009 at Year 5) as compared with CON. No effects were found for MICT. The study demonstrates that in addition to having a protective cardiovascular effect, HIIT also has a beneficial effect on muscle strength and physical performance in a relatively healthy population of older adults.
Exercise has been proposed as both a preventive and therapeutic countermeasure; however, its effectiveness across different disuse conditions and timings of implementation remains uncertain. This systematic review and meta-analysis (PROSPERO: CRD42021256599) searched ClinicalTrials.gov, Cochrane Central, PubMed, SPORTDiscus, Web of Science, Scopus, CINAHL and SciELO from inception to May 2021, with an update in March 2025. Randomized and nonrandomized controlled trials examining exercise interventions during or after muscle disuse were included according to the PICOS framework. Random-effects meta-analysis evaluated effects on muscle strength, power and mass across hospitalization, bed rest and spaceflight conditions. Effect sizes (ES) are reported as standardized mean differences with 95% confidence intervals (CI). A total of 1754 participants (66% male, 34% female; mean age 49 ± 22 years) were included. Preventive exercise interventions significantly improved muscle strength and power across disuse models. During hospitalization, exercise significantly increased muscle strength (ES = 0.60, 95% CI [0.42, 0.78]; p < 0.0001; I2 = 76%). This effect remained significant in multilevel analyses accounting for within-study dependence (ES = 0.58, 95% CI [0.19, 0.96]; p = 0.003). During bed rest, conventional random-effects analyses indicated large effects (ES = 1.16, 95% CI [0.60, 1.71]; p < 0.0001; I2 = 55%); however, multilevel models incorporating correlated lower limb strength and power outcomes showed no significant pooled effect (ES = 0.06, 95% CI [-1.31, 1.43]; p = 0.93). In contrast, spaceflight studies demonstrated small, nonsignificant effects on muscle strength and power (ES = 0.10, 95% CI [-0.32, 0.51]; p = 0.65), and pooled standardized mean change analyses indicated no significant overall change in muscle mass (ES = 0.002, 95% CI [-0.077, 0.080]; p = 0.966). When exercise was applied therapeutically after disuse, a trend toward improved muscle strength and power was observed (ES = 0.23, 95% CI [-0.01, 0.47]; p = 0.06; I2 = 12%), although multilevel models showed no significant effects (ES = 0.30, 95% CI [-0.34, 0.95]; p = 0.361). Exercise significantly preserved or increased muscle mass during bed rest (ES = 0.47, 95% CI [0.19, 0.74]; p = 0.0009) and spaceflight (ES = 0.27, 95% CI [0.05, 0.48]; p = 0.02). Exercise, particularly resistance training, attenuates muscle strength loss and preserves muscle mass during hospitalization and bed rest, whereas no consistent benefits are observed during spaceflight. Exercise initiated after disuse shows modest potential for restoring muscle function. Current evidence is insufficient to determine whether preventive or therapeutic initiation provides superior outcomes. Further high-quality randomized controlled trials are required to define the optimal timing, modality and dose of exercise for clinical rehabilitation and aerospace applications.
Peripheral nerve injury-induced muscle atrophy shares core pathophysiological features with systemic wasting disorders including cachexia and sarcopenia, yet early molecular triggers remain undefined. This study investigates the pathogenic role of receptor-interacting protein kinase 3 (RIPK3) in denervation atrophy. Sciatic denervation was induced in rats for initial time-course transcriptomics and in mice for genetic and pharmacological studies. Assessments in wild-type and RIPK3-knockout mice included transcriptomics (RNA-seq, qPCR), muscle morphology (wet weight ratio, cross-sectional area), histological inflammation (H&E, CD68 immunofluorescence), mitochondrial function (complex I/V activity, ultrastructure and biogenesis/fission regulators), STRING analysis to identify downstream effectors, validated key effectors NOX2 and NOX4 (qPCR/Western blotting) and associated redox status (DHE staining), and analysis of myofibrillar protein content and proteolytic markers (Western blotting). Confirmatory studies included RIPK3 overexpression in C2C12 myotubes and its pharmacological inhibition (GSK872) in mice. RIPK3 emerged from transcriptomic analysis as an early upregulated mediator in denervated muscle, with protein levels increasing approximately threefold at 36 h post-injury. Genetic ablation of RIPK3 attenuated muscle atrophy, as shown by improved gastrocnemius wet weight ratio (p = 0.0110). This protective effect was directly evidenced by a 40.7% increase in cross-sectional area (p = 0.04). The morphological preservation was accompanied by markedly suppressed expression of key atrophy markers, including MAFbx, MuRF1 and FoxO3a (all p < 0.01), and preserved MHC levels (p = 0.0278). Mechanistically, RIPK3 knockout reduced inflammation, enhanced oxidative phosphorylation (GSEA FDR < 0.001) and partially restored mitochondrial function, evidenced by significantly increased complex I (p = 0.0438) and complex V (p < 0.001) activity, preserved ultrastructure, upregulated PGC-1α and NRF2 (both p < 0.05) and downregulated mitochondrial fission proteins (p-DRP1, MFF, FIS1; all p < 0.01). STRING analysis predicted NOX4 as a key downstream effector, validated by reduced NOX4 protein (-46.6%, p = 0.0366) and a consequent 52.2% decrease in ROS accumulation (p < 0.001). Consistently, RIPK3 overexpression in C2C12 myotubes elevated NOX4 (p = 0.0046) and atrophy markers, whereas pharmacological inhibition of RIPK3 in mice replicated the protective phenotype, increasing muscle wet weight ratio (p = 0.0277) and suppressing NOX4 (p = 0.0398) and proteolytic markers. Denervation activates RIPK3 as a master regulator that drives muscle atrophy via NOX4/ROS-induced mitochondrial dysfunction, sustained inflammation and ubiquitin-proteasome activation. Targeting RIPK3 preserves muscle mass and may offer a novel therapeutic strategy for neurogenic muscle atrophy, with possible implications for related wasting disorders.
Diabetes mellitus-a chronic metabolic disorder associated with an increased risk of muscle atrophy-can significantly impact patients' quality of life and overall health outcomes. While antidiabetic medications are crucial for managing blood glucose levels, some have been linked to muscle-related adverse events, potentially exacerbating the already elevated risk of muscle deterioration in diabetic patients. However, a comprehensive analysis of muscle atrophy-related adverse events across different classes of antidiabetic drugs has been lacking. Therefore, this study investigates the profile of muscle atrophy-related adverse events across major antidiabetic drug classes using the World Health Organization's (WHO's) Individual Case Safety Reports database. A pharmacovigilance analysis was conducted using data from VigiBase, the WHO's global reporting database, from 1968 to September 2025. The study examined adverse event signals related to muscle atrophy, sarcopenia, muscular weakness and motor function decline for nine classes of antidiabetic medications. Reporting odds ratios (RORs) were calculated to assess signal detection, and co-occurrence patterns of adverse events were analysed. Among 41 551 306 adverse event reports, 2 095 847 were related to antidiabetic medications. Safety signals for muscle atrophy were detected with sulfonylureas (ROR: 1.2, 95% CI: 1.01-1.43, p = 0.042), GLP-1 analogues (ROR: 1.2, 95% CI: 1.02-1.41, p = 0.031) and SGLT2 inhibitors (ROR: 1.5, 95% CI: 1.19-1.78, p < 0.001). SGLT2 inhibitors also showed a signal for sarcopenia (ROR: 6.2, 95% CI: 3.71-10.3, p < 0.001). Biguanides demonstrated signals for muscular weakness (ROR: 1.6, 95% CI: 1.54-1.71, p < 0.001) and motor function decline (ROR: 1.7, 95% CI: 1.41-2.13, p < 0.001). Thiazolidinediones, glinides, DPP-4 inhibitors and alpha-glucosidase inhibitors showed no safety signals for the examined adverse events. Additionally, co-occurrence analysis revealed frequent associations between muscle atrophy and nausea/vomiting, falls and decreased appetite across different drug classes. These findings indicate notable differences in the profiles of muscle atrophy-related adverse events among major classes of antidiabetic drugs, suggesting that drug selection may influence the risk of muscle function decline in patients. Clinicians should consider these safety profiles when prescribing antidiabetic therapies; however, causal relationships cannot be inferred solely from pharmacovigilance data. Further studies are warranted to establish causality between antidiabetic drug use and muscle-related adverse events and to elucidate the underlying mechanisms.
Prader-Willi syndrome (PWS) is characterized by sarcopenic obesity; however, validated screening tools for muscle mass in clinical settings are lacking. This study aimed to evaluate the diagnostic utility of muscle ultrasound (US) for detecting low muscle mass in individuals with PWS. This observational, cross-sectional study recruited 48 individuals with genetically confirmed PWS (International Classification of Diseases, 10th Revision Code Q87.1) from a specialized clinic (October 2022-June 2024). Appendicular skeletal muscle index (ASMI) via dual-energy x-ray absorptiometry (DXA) served as the primary outcome. Sarcopenia was defined based on Asian Working Group for Sarcopenia (AWGS) 2019 criteria. US predictors included muscle thickness (MT) of the rectus femoris (RF), vastus lateralis and gastrocnemius medialis (GM); pennation angle; and RF cross-sectional area (CSA). Analysis included Spearman's correlation (ρ), multivariable linear regression (B) and receiver operating characteristic (ROC) curves. The cohort (n = 48; 47.9% women; median age 19.5 years, interquartile range [IQR] 12.3-26.8) had a 100% prevalence of obesity (median BMI 27.1 kg/m2, IQR 22.2-31.4). All participants (100%) had a history of growth hormone treatment; 62.5% exhibited the deletion subtype. Low muscle mass was observed in 60.4% (n = 29), confirmed sarcopenia in 52.1% (n = 25) and severe sarcopenia (including low physical performance) in 20.8% (n = 10) participants. GM MT showed the strongest correlation with ASMI (ρ = 0.689, p < 0.001, 95% confidence interval [CI] 0.50-0.81), followed by RF CSA (ρ = 0.587, p < 0.001). Multivariable regression identified GM MT as a significant independent predictor of ASMI (B = 0.957, p = 0.011) after adjusting for BMI (B = 0.128, p < 0.001), age (B = 0.020, p = 0.044) and sex (B = -0.376, p = 0.029; R2 = 0.67). ROC analysis for detecting low muscle mass yielded an area under the curve for GM MT of 0.759 (95% CI 0.616-0.901, p = 0.003), with an optimal cut-off of 1.69 cm (sensitivity 86.2%, specificity 63.2%). Sarcopenia affects 52.1% of patients with PWS, with 20.8% meeting criteria for severe sarcopenia. Ultrasound of the gastrocnemius medialis is a valid, radiation-free predictor of skeletal muscle mass and serves as a practical diagnostic tool for sarcopenic obesity in PWS.
Obesity and diabetes impair the ability of the muscle to regenerate, repair and remodel, resulting in a gradual decrease in muscle mass and function. However, the underlying mechanisms and effective therapeutic strategies remain poorly understood. M2 macrophages within skeletal muscle play an important role in tissue recovery following injury. This study aims to investigate the role of M2 macrophages derived transforming growth factor-beta 1 (Tgf-β1) in regulation of skeletal muscle function under diet-induced obese conditions. An CD206+ M2 macrophage-specific Tgf-β1 gene knockout (Tgf-β1 KO) mouse model was generated by crossing CD206-CreERT2 mice with Tgf-β1f/f mice, followed by tamoxifen administration to induce Tgf-β1 gene deletion. Mice were then placed on a high-fat diet (HFD) for 12 weeks to develop obesity-induced skeletal muscle dysfunction. The study used multiple physiological and molecular analyses, including exercise tolerance, hanging time, grip strength, glucose and insulin tolerance tests, western blotting, RT-qPCR and others. The present findings demonstrated improved exercise performance, as evidenced by increased running distance twice (p = 0.0008) in Tgf-β1 KO mice. Deletion of CD206+ M2 macrophage-specific Tgf-β1 stimulates fibro-adipogenic progenitors (FAPs), inducing Follistatin (Fst) expression by 1.70-fold (p = 0.04) and follistatin-like protein 1 (Fstl1) by 2.60-fold (p = 0.01) in tibialis anterior (TA), thus enhancing myogenesis. The Tgf-β1 KO mice showed increased muscle fibre type I (Myh7 by 2.40-fold, p = 0.005), muscle fibre type IIa (Myh2 by 1.50-fold, p = 0.003), type IIx (Myh1 by 2.35-fold, p = 0.002) in soleus and type II (Myh4 by 1.76-fold, p = 0.02) in TA. In Tgf-β1 KO mice, insulin-stimulated Akt phosphorylation was significantly increased in adipose tissue by 2.14-fold (p = 0.0003) and in the liver by 1.62-fold (p = 0.01). Besides, the Tgf-β1 KO mice showed increased circulating adiponectin by 1.23 fold (p = 0.003), thereby activating the AMPK/SIRT1/PGC-1α pathway with the phosphorylation level of AMPKα increased by 1.5-fold (p = 0.02), and PGC1α protein level increased by 2.2-fold (p = 0.02) via increased AdipoR1 mRNA expression by 1.8-fold in TA, p = 0.0001 and by 1.8-fold in soleus, p = 0.01 in skeletal muscle, leading to improved mitochondrial function in skeletal muscle. The CD206+ M2 macrophage-specific Tgf-β1 deletion ameliorates obesity-induced muscle dysfunction, potentially via two distinct mechanisms. Firstly, it enhances myogenesis by promoting FAP-mediated expression of Fst and Fstl1, thereby augmenting myogenesis-related gene expression in skeletal muscle. Secondly, it also induces adipocytes to produce and secrete adiponectin into the bloodstream, thereby enhancing mitochondrial function via the AMPK/SIRT1/PGC-1α pathway.