IntroductionSarcopenia is a debilitating, age-associated condition characterized by the progressive loss of skeletal muscle mass, strength, and functional capacity. It is a major contributor to frailty, loss of independence, metabolic disorders, and increased mortality in the elderly population.1,2,3 With the rapid expansion of aging societies worldwide, sarcopenia has emerged as an important public health concern with substantial clinical and socioeconomic consequences, imposing a growing burden on healthcare systems and significantly reducing the quality of life of affected individuals. The prevalence of sarcopenia continues to increase with advancing age, contributing to compromised mobility, falls, fractures, disability, and prolonged hospitalization, thereby imposing a substantial burden on healthcare system. Consequently, preserving skeletal muscle mass and metabolic function has become a major priority for promoting healthy aging and extending health span. However, despite extensive research efforts, the molecular mechanisms responsible for age-related muscle deterioration remain incompletely understood, and effective pharmacological therapies for sarcopenia are still lacking,4,5 highlighting the urgent need to identify novel therapeutic targets and effective intervention strategies for preventing disease progression.Mitochondrial dysfunction and disrupted lipid metabolism are increasingly recognized as central drivers of sarcopenia.6,7,8,9,10 Excessive intramyocellular lipid accumulation, reduced oxidative capacity, impaired insulin sensitivity, and decreased mitochondrial content and function in aging skeletal muscle are consistently observed and are strongly associated with progressive muscle degeneration during aging.11,12,13,14 These metabolic abnormalities contribute to energy deficiency, excessive reactive oxygen species production, and the activation of catabolic pathways, collectively promoting muscle weakness and functional decline.15,16,17,18 Accumulating evidence further indicates that impaired mitochondrial quality control, including defects in mitochondrial biogenesis, dynamics, and mitophagy, accelerates the deterioration of muscle metabolic capacity during aging. Failure to maintain mitochondrial integrity disrupts ATP production and increases oxidative damage, creating a vicious cycle that exacerbates cellular dysfunction and muscle atrophy. Importantly, lipid metabolic disturbances and mitochondrial dysfunction are closely interconnected, as impaired fatty acid utilization can exacerbate oxidative stress and further compromise mitochondrial homeostasis. Conversely, defective mitochondrial oxidative phosphorylation limits lipid oxidation, leading to the accumulation of toxic lipid intermediates that further impair insulin signaling and metabolic flexibility in skeletal muscle. Several molecular regulators play pivotal roles in maintaining these processes in skeletal muscle. Among them, the mitochondrial deacetylase SIRT3 and the transcriptional coactivator PGC-1α are critical regulators of mitochondrial biogenesis, oxidative metabolism, and cellular stress resistance.19,20,21,22,23 Together these regulators coordinate mitochondrial adaptation to energetic stress and are essential for preserving metabolic homeostasis and muscle function during the aging process.In aging muscle, reduced expression of SIRT3 and PGC-1α has been associated with defective mitochondrial regulation, impaired oxidative stress responses, and abnormal lipid metabolism, ultimately contributing to lipid accumulation and metabolic dysfunction.23,24,25 Consistent with these observations, studies in sarcopenic muscle have shown that mitochondrial dysfunction is accompanied by the suppression of anabolic signaling pathways and the development of anabolic resistance.15,26,27 In addition to mitochondrial regulators, liver X receptors (LXRs), members of the nuclear receptor family, are key regulators of lipid and cholesterol metabolism.28 While the liver X receptor α (LXRα) predominantly regulates hepatic lipid homeostasis, LXRβ seems to play a dominant role in skeletal muscle.29,30 LXRs form heterodimers with other nuclear receptors, such as Retinoic X Receptors (RXRs) or Peroxisome Proliferator-Activated Receptors (PPARs), to modulate genes involved in lipid metabolism, glucose utilization, and energy homeostasis.31,32,33 Beyond their metabolic functions, LXR agonists have demonstrated anti-inflammatory and cytoprotective effects in several disease models, including atherosclerosis,33,34,35 suggesting broader roles in maintaining cellular homeostasis and protecting tissues from metabolic stress under pathological and age-related conditions. Emerging evidence further suggests that LXR signaling may influence mitochondrial quality control and oxidative metabolism through functional interactions with transcriptional regulators involved in energy homeostasis. However, the mechanisms linking lipid sensing to mitochondrial regulation in aging skeletal muscle remain poorly understood, and the potential contribution of LXRβ to this process has not been established. This knowledge gap has limited the development of mechanism-based therapeutic strategies aimed at restoring metabolic homeostasis and mitochondrial function in aging skeletal muscle. Therefore, identifying the molecular mechanisms by which LXRβ coordinates lipid metabolism and mitochondrial function may provide new opportunities for developing targeted therapies to combat sarcopenia and other age-related metabolic disorders.Our previous study demonstrated that Inonotus obliquus (I. obliquus) extract improves muscle regeneration and attenuates DEX-induced muscle atrophy in association with the modulation of PGC-1α and mitochondrial-related pathways.36 In the present study, we identified Ino, a major mycosterol component of I. obliquus, as a key bioactive compound that mediates these effects. We therefore investigated the effects of Ino on muscle metabolism, mitochondrial regulation, and muscle performance during aging. Our findings reveal that Ino alleviates muscle degeneration, enhances mitochondrial biogenesis and oxidative capacity, and mitigates lipid accumulation in aged skeletal muscles. Furthermore, we identify LXRβ as a previously unrecognized regulator of mitochondrial homeostasis and show that Ino exerts its protective effects through activation of the LXRβ/SIRT3/PGC-1α signaling pathway. Collectively, these results highlight Ino as a promising therapeutic candidate for combating age-related sarcopenia and other muscle wasting conditions.ResultsMuscle aging is associated with metabolic disturbances, mitochondrial dysfunction, and a decrease in the activity of the SIRT3/PGC-1α pathwayTo gain insights into metabolic changes associated with muscle aging, young (Y) and aged (A) mouse muscles were subjected to metabolite profiling (Fig. 1a). Hierarchical clustering revealed aging-related alterations in diverse metabolites. Among the intramuscular metabolites, those related to fatty acid or amino acid metabolism were markedly altered, suggesting disturbed amino acid or fatty acid homeostasis.Fig. 1Full size imageMuscle aging is associated with metabolic disturbances, mitochondrial dysfunction, and a decrease in the activity of the SIRT3/PGC-1α pathway. a Untargeted metabolomic profiling of young and aged mouse skeletal muscle reveals age-associated shifts in metabolite abundance. The heatmap displays z score-scaled metabolite levels, and the side annotation indicates chemical classes. b Heatmap showing the GSEA-based metabolic pathways across the myonuclei. The color denotes the normalized enrichment score. c Heatmap showing the expression profiles of oxidative stress-related genes across myonuclei. Red arrows indicate the most prominent changes in expression patterns. d Quantitative analysis of myofiber cross-sectional area from H&E-stained sections in a human cohort derived from the GTEx database using a computational pipeline that measures fiber size in pixel units and converts values to micrometers using the recorded pixel size. Whole-slide images were acquired with recorded pixel sizes, and regions of interest were digitally magnified fourfold while the physical scale was preserved. Scale bar = 180 µm. e qRT‒PCR analysis of mRNAs related to mitochondrial metabolism in the gastrocnemius (GAS) muscles of 3-month-old (young) and 24-month-old (aged) male mice. Rn18s was used as an internal control. (n = 4 for young; n = 3 for aged). f Immunoblot analysis of PGC-1α and SIRT3 in total lysates or mitochondrial (Mito) fractions of GAS muscles from young and aged male mice. The quantification of the relative protein expression levels is shown on the right. Ponceau S or VDAC1 served as loading controls (n = 3 per group for total lysates; n = 4 for young mito; n = 3 for aged mito). g Relative ratio of mtDNA (Mt-co2) to nuclear DNA (nDNA, Gapdh) in GAS muscles from young and aged male mice (n = 4 per group). h Relative integrated density (IntDen) ratio of JC-1 aggregates (red) to monomers (green) in single myofibers isolated from the EDL muscles of young and aged male mice (n = 4 for young; n = 3 for aged; 3–6 myofibers analyzed per individual). Representative images are shown in Supplementary Fig. 1e. i Relative IntDen for mitoSOX fluorescence (red) in single myofibers isolated from the EDL muscles of young and aged male mice. Representative images are shown in Supplementary Fig. 1f (n = 4 for young; n = 3 for aged; 3–4 myofibers analyzed per individual). The data are expressed as the means ± SDs. Statistical significance was determined using an unpaired two-tailed Student’s t test. *p