GRAPHICAL ABSTRACT

INTRODUCTION

Catecholamines, including epinephrine, norepinephrine, and dopamine, are hormones that play a vital role in regulating physiological processes in the body [1,2]. As key regulators of body composition, they promote muscle growth and reduce fat mass via the activation of the β-adrenergic receptor [3,4], suggesting that they could have potential therapeutic applications as β-adrenergic agonists for the treatment of various conditions, such as muscle wasting and obesity [5–7]. However, high levels of catecholamines can lead to muscle wasting through chronic overstimulation of the β-adrenergic receptor [8]. Moreover, prolonged elevation of catecholamine levels increases metabolic demands and oxidative stress in muscle tissue, contributing to catabolic processes and disrupting the balance between muscle protein synthesis and degradation, favoring muscle atrophy [9,10]. While catecholamines generally support muscle growth by stimulating myogenesis and muscle protein synthesis, their prolonged elevation, as seen in conditions such as pheochromocytoma and the chronic stress associated with intensive care units, can induce muscle wasting. This phenomenon is attributed to persistent β-adrenergic receptor activation, which promotes oxidative stress and protein breakdown, overriding the anabolic effect of catecholamines [11]. Although these effects are well documented in overt catecholamine excess, such as in pheochromocytoma, the physiological significance of mild but measurable elevations that do not meet diagnostic thresholds remains unclear. In this study, we use the term ‘high-normal catecholamine levels’ to describe a state in which plasma metanephrine and normetanephrine levels are elevated relative to the general population but remain below the clinical cutoff for pheochromocytoma diagnosis (i.e., below twice the upper limit of normal). This operational definition reflects a state of sustained catecholaminergic activity that may still affect muscle homeostasis in the absence of overt disease. Despite the known physiological effects of catecholamines, the impact of normal physiological variations in catecholamine levels on muscle mass remains poorly understood. Further research is needed to elucidate how variations in catecholamine concentrations within the normal range influence muscle physiology and overall muscle mass.

Demographic factors such as age and sex may also alter catecholamine levels and the physiological effects of catecholamines in humans [12,13]. Analysis of urine catecholamine metabolites indicates that women excrete epinephrine at lower levels than men [14]. Sex also governs the intensity of the catecholamine response to acute decreases in blood glucose [15]. Moreover, higher sympathetic activity in women with identical exercise workloads has been reported to be due to their relatively smaller skeletal muscle mass compared with men [16,17]. On the other hand, catecholamine synthesis, in general, declines with the aging of the adrenal glands in mice [18]. By contrast, plasma catecholamine levels are higher in old men aged 70–92 years than in young men aged 19–28 years [19]. Moreover, catecholamine sensitivity decreases with age and is mediated by β1-adrenergic receptors, resulting in impaired cyclic adenosine monophosphate (cAMP) production by adenylate cyclase [20]. Thus, understanding these variations in catecholamine levels is crucial for interpreting their physiological implications across diverse patient populations.

In this study, we explored the influence of physiological variations in catecholamine levels on muscle mass using clinical data and advanced imaging analysis. Artificial intelligence (AI)-assisted analysis of abdominal computed tomography (CT) images was used to evaluate body composition, while clinical data provided information on catecholamine concentrations. Furthermore, muscle cell experiments were conducted to investigate the downstream signaling pathways of β-adrenergic receptor activation and their effects on myogenesis and muscle differentiation. This comprehensive approach aims to deepen our understanding of the interplay between catecholamines and muscle physiology, with potential implications for clinical management strategies aimed at preserving muscle health.

METHODS

Study population

This retrospective study examined 830 patients diagnosed with adrenal incidentalomas over a period of 10 years from August 2013 to March 2024 at Chungnam National University Hospital in Daejeon. The cohort included 415 males and 415 females, aged between 16 and 91 years. Initially, 1,168 patients with adrenal incidentalomas were identified from the hospital’s electronic medical records. However, 160 patients were excluded because their plasma catecholamine fraction, represented by metanephrine and normetanephrine levels, the most direct diagnostic indicators of pheochromocytoma, were two-fold above the upper limit of normal (metanephrine, 0–0.49 nmol/L; normetanephrine, 0–0.89 nmol/L) or because they were followed up at another hospital. As the study aimed to investigate the partial correlation between catecholamines and muscle indices, patients with abnormally high plasma catecholamine levels, 89 patients with a history of pheochromocytoma surgery, and patients with missing CT data and body mass index (BMI) data were excluded. Fifteen patients with other adrenal diseases, such as Cushing’s disease and primary aldosteronism, were also excluded, resulting in 830 patients being included in the study. Some of the patients diagnosed with pheochromocytoma did not undergo surgery at the study hospitals or were lost to follow-up (Supplemental Fig. S1).

Among the remaining patients, those with plasma metanephrine and normetanephrine concentrations within or mildly above the reference range (metanephrine, 0–0.49 nmol/L; normetanephrine, 0–0.89 nmol/L), but below twice the upper limit of normal, were classified as having high-normal catecholamine levels for the purpose of this study. This definition is intended to capture individuals with persistent but non-pathological catecholaminergic activity, distinct from both pheochromocytoma and fully normal levels.

Laboratory measurements

This retrospective study utilized the following variables for analysis: age, sex, height, weight, BMI, β-blocker use, and CT scans. In addition, baseline clinical parameters such as blood pressure and antihypertensive medication status were reviewed to account for potential confounding effects on catecholamine metabolism. Blood tests included renal function markers such as blood urea nitrogen, creatinine, and estimated glomerular filtration rate (eGFR); and total protein and albumin levels. Endocrine parameters included plasma renin and aldosterone levels to assess hormonal influences on muscle and fat metabolism. In addition, laboratory assessments included plasma metanephrine and normetanephrine levels, the overnight dexamethasone suppression test (ODST), and 24-hour urinary free cortisol (24h UFC). Blood levels of metanephrine and normetanephrine in nmol/L were collected rather than 24-hour urine levels. Given that either a dexamethasone suppression test or a 24h UFC test is mandatory after adrenal incidentaloma diagnosis, the results of these tests were collected to further explore their association with adiposity. CT scans, which are frequently ordered for adrenal incidentaloma patients to screen for other conditions, were used to determine the size and location of adrenal tumors (bilateral or unilateral). AI was employed to analyze CT data and measure total abdominal muscle area (TAMA), low-attenuation muscular area (LAMA), normal-attenuation muscular area (NAMA), intermuscular adipose tissue (IMAT), and skeletal muscle area (SMA).

The study first analyzed the relationship between age and plasma metanephrine and normetanephrine levels, and then analyzed the relationship between age and muscle indices. Finally, the partial correlation between plasma catecholamines and CT-derived muscle indices was examined by correlating plasma catecholamines levels with quantitative muscle measurements. Patient registration numbers and names were de-identified and anonymized, and the study was approved by the Medical Research Ethics Review Board, approval number 2019-06-063. Written informed consent by the patients was waived due to a retrospective nature of our study.

CT image acquisition

A standardized CT acquisition protocol was used for health checkups. Abdomen and pelvis CT examinations were performed using the Somatom Definition (Siemens Healthineers, Erlangen, Germany), Discovery CT750 HD (GE Healthcare, Milwaukee, WI, USA), or LightSpeed VCT scanner (GE Healthcare). All image data were reconstructed with a slice thickness of 5 mm using the filtered back-projection technique with the soft tissue reconstruction algorithm (B30f kernel, Siemens Healthineers; standard kernel, GE Healthcare). For contrast enhancement, 100–150 mL of iopromide (Ultravist 370 or Ultravist 300, Bayer Schering Pharma, Berlin, Germany) was intravenously administered using an automatic power injector at an injection speed of 2.5–3 mL/sec. A fixed scan delay of 70 seconds after contrast agent injection was used for CT acquisition.

Assessment of SMA and quality

ClariMetabo utilizes two deep learning models to quantify abdominal fat and muscle components in CT images. The first model, trained on maximum intensity projected CT images from over 900 patient scans, was devoted to localizing the vertebra body (T12–L4), and achieved 99.1% categorical accuracy. Upon identifying the L3 level, the second model, a classical 2D UNet, trained with over 40,000 CT images, automatically segmented abdominal fat and muscle, achieving a dice score of 0.96–0.98. The measurements provided the area and mean CT-attenuation (Hounsfield unit [HU]) for fat and muscle tissues, including IMAT, LAMA, NAMA, SMA, and TAMA. The subcutaneous and visceral fat regions were determined in the −240 to −10 HU range. NAMA, LAMA, and IMAT were defined by HU ranges 30 to 150, −29 to 29, and −190 to −30, respectively. The SMA is the sum of LAMA and NAMA, while TAMA includes IMAT, LAMA, and NAMA. Using the patient’s height and weight information, ClariMetabo was used to calculate BMI, and myosteatosis percentage defined as 100×(1−NAMA/TAMA). All these imaging biomarkers were previously reported to be predictive of metabolic risk, sarcopenia, and myosteatosis (Fig. 1).

Chemicals and reagents

β-Adrenergic blockers refer to drugs that selectively block β-adrenergic receptors among adrenergic receptors in the sympathetic nervous system. These drugs are also known as β-blockers or β-adrenergic antagonists and are clinically used to regulate arrhythmias or prevent secondary myocardial infarction. In this study, Atenolol was selected as a representative drug, while clenbuterol was used as an agonist. Additionally, LY3214996 was employed to inhibit the extracellular signal-regulated kinase (ERK) signaling pathway.

Cell culture and induction of differentiation

The C2C12 mouse-derived cell line (American Type Culture Collection, CRL-1772; Manassas, VA, USA) was cultured at 37°C in a 5% CO₂ atmosphere. During the proliferation phase, cells were maintained in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) (Welgene, Gyeongsan, Korea; LM001-05) supplemented with 10% fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA; 16000044) and 1% penicillin/streptomycin. To investigate the signaling pathway, C2C12 cells were seeded into a 24-well plate, and serum starvation was performed for 4 hours at 80% confluency before reagent treatment.

The muscle creatine kinase (MCK) promoter-driven enhanced green fluorescent protein (EGFP) reporter C2C12 (MCK::EGFP C2C12) cell line was used to visualize and quantify myogenesis and myotube formation. The MCK promoter, known for its muscle-specific activity, drives GFP expression exclusively in differentiated myotubes. For differentiation, cells were seeded into a 96-well plate and the medium was replaced with DMEM supplemented with 5% horse serum (HS) and 1% penicillin/streptomycin. The medium was refreshed every 2 days throughout the experimental period. Human primary myoblast cells obtained from Korea Research Institute of Bioscience and Biotechnology (KRIBB, P01-201708-31-004) were cultured in Ham’s F-10 cell culture media (Gibco, 11550043) supplemented with 1% penicillin/streptomycin, 20% FBS, and basic fibroblast growth factor (50 ng/mL).

Western blot analysis

C2C12 cells were treated with clenbuterol, a β-adrenergic receptor agonist, with 10-fold serial concentrations from 1 nM to 1 μM consistent with prior in vitro studies [8,21] to examine the effects of high-normal β-adrenergic stimulation. Additionally, selected β-blocker, atenolol, was applied at 0.1 or 1 μM concentrations, comparable to the concentrations used for clenbuterol. Treatments were performed for 10 or 30 minutes. Western blot analysis was used to investigate the expression of proteins belonging to the β-arrestin pathway in C2C12 cells. Cells were rinsed with cold phosphate-buffered saline and lysed with radioimmunoprecipitation assay (RIPA) buffer (LPS Solution, Daejeon, Korea; CBR002) supplemented with 0.1 mM Na₃VO₄, 1 mM NaF, 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF), 5 mg/mL aprotinin, and sodium dodecyl sulfate (SDS). Protein samples were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on a 10% gel and transferred onto a nitrocellulose membrane. The membrane was blocked with 5% skim milk (for ERK, protein kinase A [PKA], and β-actin) or 5% bovine serum albumin (BSA) (for phospho-ERK and phospho-Protein kinase A catalytic subunit [PKAc]) at room temperature for 30 minutes. Primary antibodies diluted 1:1,000 in 5% skim milk or 5% BSA were added to the membrane and incubated overnight at 4°C. After three washes with 1× Tris-buffered saline with Tween-20 (TBST) buffer (Trisbuffered saline with 0.1% Tween 20, pH 7.5) for 30 minutes each, the membrane was incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 hour. Following three additional washes with 1× TBST, protein bands were visualized using enhanced chemiluminescence solution and quantified using the ChemiDoc imaging system (Thermo Fisher, Waltham, MA, USA; iBright CL1500). Primary antibodies used included phospho-PKAc (Cell Signaling Technology, Danvers, MA, USA; 5661), PKA (Cell Signaling Technology, 5842), phospho-ERK (Cell Signaling Technology, 4377), ERK (Santa Cruz Biotechnology, sc-94), phospho-AKT (Cell signaling Technology, 9271), AKT (Cell signaling Technology, 4691), phospho-p38 (Cell signaling Technology, 9211), p38 (Cell signaling Technology, 9212), phospho-forkhead box O3a (phospho-FOXO3a; Cell signaling Technology, 14724), FOXO3a (Cell signaling Technology, 12829), phospho-FOXO1 (Cell signaling Technology, 9461), FOXO1 (Cell signaling Technology, 2880), and β-actin (sc-47778) as a loading control.

Morphometric analysis

To evaluate myogenesis and myotube formation, the Incucyte Live-Cell Analysis System (Sartorius, Göttingen, Germany) was employed. MCK::EGFP C2C12 cells were seeded into a 96-well plate (SPL, Pocheon, Korea; 30096) and differentiated in DMEM supplemented with 5% HS under confluent conditions. Differentiated cells were then transferred to the Incucyte Live-Cell Imaging System. Phase-contrast and GFP images were captured daily throughout the experimental period. Using Incucyte’s integrated image analysis software, GFP signals were quantified by calculating the total green object area (μm²). All experiments were conducted with 12 replicates to ensure reproducibility and statistical reliability. For immunostaining, differentiated C2C12 cells were fixed in 4% formaldehyde for 15 minutes and permeabilized in 0.2% Triton X-100 for 15 minutes. Samples were blocked for 30 minutes with 2% BSA in TBST, and incubated with anti-myosin heavy chain (MyHC; Santa Cruz Biotechnology, sc-376157) overnight. After washing samples with TBST, AlexaFluor 488 (Invitrogen, Waltham, MA, USA; A21121) secondary antibody was added for 30 minutes, and then 4′,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific, 62248) staining was performed and images were captured. For the measurement of myotube diameter and fusion index, four images were randomly selected. The diameters of MyHC-positive myotubes containing ≥3 nuclei were quantified in each image (n=10 per image). The fusion index was calculated as the ratio of nuclei within MyHC-positive myotubes containing ≥3 nuclei to total nuclei in each image. The GFP signals of 16 fields in each sample were quantified by calculating the total green object area (μm²) using Incucyte’s integrated image analysis software.

Statistical analysis

Variable characteristics are presented as mean±standard deviation or median and interquartile range for variables that are not normally distributed. Statistical analyses were performed using SPSS version 30 (IBM Co., Armonk, NY, USA). Partial correlation analyses were performed between plasma metanephrine, normetanephrine, and CT-derived muscle indices, controlling for age, β-blocker use, eGFR, albumin, total protein, blood pressure, renin, aldosterone with 24h UFC, and ODST. In addition, sex-specific differences in catecholamine distributions were examined using unpaired two-tailed t tests. Correlogram and violin plots were generated in R version 4.4.0 (R Foundation for Statistical Computing, Vienna, Austria) using the ggplot2 package. Plasma metanephrine and normetanephrine levels were each categorized into tertiles, and muscle indices were compared across groups using one-way analysis of variance (ANOVA). Data normality was checked before analysis, and results were plotted using Prism software version 8.3 (GraphPad Software Inc., San Diego, CA, USA). To assess statistically significant changes in metanephrine and normetanephrine levels with increasing age, subjects were divided into age groups, those above 60 years of age and those below 60 years of age, and t tests were performed for both metanephrine and normetanephrine levels. The total green object area in MCK::EGFP C2C12 cells following clenbuterol treatment was shown as mean±standard error of mean (SEM), and P values were calculated by two-way ANOVA with Dunnett’s multiple comparison test. Myotube diameter, total green object area, and fusion index in C2C12 cells were shown as mean±SEM, and P values were calculated by an ordinary one-way ANOVA with Dunnett’s multiple comparison test. A P value of less than 0.05 was considered statistically significant.

RESULTS

Study population

This study aimed to investigate the partial correlation between catecholamine levels and muscle mass. The study population comprised 830 participants (415 males and 415 females) with a mean age of 59.36±11.73 years and BMI of 24.99±3.57 kg/m². AI-assisted comprehensive body composition analysis was used to measure specific fat areas, including visceral fat area (VFA; 151.83±79.80 cm²), subcutaneous fat area (145.71±64.09 cm²), and total fat area (TFA; 297.54±114.35 cm²), alongside specific muscle compositions, including LAMA (35.94±13.15 cm²), NAMA (82.60±32.09 cm²), TAMA (128.81±30.53 cm²), and SMA (118.81±30.53 cm²), while catecholamine levels were measured in terms of metanephrine (0.15±0.09 nmol/L) and normetanephrine (0.58±0.27 nmol/L) concentrations (Supplemental Table S1). Significant sex differences were observed in plasma catecholamine levels, with males exhibiting higher median values than those of females for both metanephrine and normetanephrine (Supplemental Table S2). These sex-specific variations in catecholamine levels were statistically significant (P<0.001). Consistent with these results, similar patterns were observed in the violin plots (Supplemental Fig. S2).

Furthermore, variations in muscle composition were also evident across age groups. TAMA, NAMA, and SMA were significantly higher in individuals aged 60 years or younger than in those aged 60 years or older regardless of sex, whereas LAMA was more frequently observed in those over 60 years of age (Supplemental Fig. S3). In addition, catecholamine levels were further categorized into tertiles (Q1–Q3), where Q1, Q2, and Q3 represented the lowest, middle, and highest tertiles of catecholamine concentrations, respectively. Sex-stratified analyses revealed consistent gradients in muscle indices across these tertiles, particularly in NAMA, SMA, and TAMA, with lower catecholamine groups (Q1) generally exhibiting higher muscle indices (Table 1). This tertile-based approach provided complementary evidence supporting the findings from the continuous partial correlation analyses.

Association between catecholamine levels and body composition

The partial correlation analysis between catecholamines, body composition, and age showed distinct sex-specific patterns. In men, metanephrine levels were significantly and inversely correlated with VFA, TFA, LAMA, SMA, and TAMA. Normetanephrine levels were negatively correlated with VFA, TFA, NAMA, SMA, and TAMA. In women, metanephrine was inversely associated with VFA, TFA, myosteatosis percentage, LAMA, SMA, and TAMA. By contrast, normetanephrine showed no significant correlations with muscle parameters in women (Fig. 2). More specifically, in men, metanephrine showed significant negative correlations with VFA (r=−0.25, P=0.004), TFA (r=−0.23, P=0.007), LAMA (r=−0.20, P=0.022), SMA (r=−0.22, P= 0.011), and TAMA (r=−0.24, P=0.006), while normetanephrine correlated negatively with VFA (r=−0.25, P=0.004), TFA (r= −0.23, P=0.010), NAMA (r=−0.18, P=0.037), SMA (r=−0.30, P<0.001), and TAMA (r=−0.31, P<0.001). In contrast, in women, metanephrine was inversely associated with VFA (r=−0.22, P=0.006), TFA (r=−0.25, P=0.002), myosteatosis percentage (r=−0.22, P=0.005), LAMA (r=−0.31, P<0.001), SMA (r= −0.16, P=0.048), TAMA (r=−0.21, P=0.010). These findings underscore the importance of evaluating sex differences when examining the metabolic and physiological impacts of catecholamines.

In this cross-sectional analysis, four distinct muscle area parameters were assessed using CT: TAMA, NAMA, LAMA, and SMA. The image shown in Fig. 3 shows a comparison of the effects of differences in catecholamine levels on NAMA and TAMA in two men with nearly identical BMIs. The red area represents NAMA and the orange area represents lipid-rich muscle or LAMA. The study stratified subjects by both sex and age (<60 years vs. ≥60 years), revealing statistically significant differences (P<0.001) across all measured parameters. Both male and female subjects demonstrated consistent age-related patterns, with the ≥60 age cohort exhibiting decreased TAMA, NAMA, and SMA values compared with their younger counterparts. Notably, LAMA was paradoxically higher in the older age group irrespective of gender. The observed values, measured in cm², were consistently higher in males than in females across all parameters, suggesting sex-specific variations in muscle composition (Supplemental Fig. S3). These findings provide quantitative evidence of age-associated alterations in muscle composition and suggest potential sexual dimorphism in the progression of sarcopenia, warranting further investigation into the underlying mechanisms of these sex-specific differences in muscle aging.

The inhibitory effect of a β-adrenergic agonist on myogenesis

Clinical data demonstrated an inverse association between catecholamines (metanephrine and normetanephrine) and SMA. To investigate this relationship mechanistically, we applied clenbuterol, a selective β2-adrenergic agonist, in vitro to assess the impact of excessive β2-adrenergic stimulation on myogenic differentiation, given that β2-adrenoceptors are the predominant subtype in skeletal muscle [3]. To visualize myotube formation and quantify GFP expression driven by the MCK promoter during differentiation, we utilized MCK::EGFP C2C12 cells (Fig. 4A). To examine the role of clenbuterol in myogenesis, MCK::EGFP C2C12 cells were treated with clenbuterol from the onset of differentiation (Fig. 4B). A dose-dependent inhibitory effect on myogenesis was observed beginning 2 days after clenbuterol treatment (Fig. 4C, D), with a significant reduction in GFP-expressing myotubes that persisted through to terminal differentiation (Fig. 4E). To assess whether clenbuterol also impairs myotube formation at a later stage of myogenesis, clenbuterol was applied to differentiated myotubes (Supplemental Fig. S4A). After 2 days of treatment, a significant reduction in GFP-expressing myotubes was observed, indicating that clenbuterol inhibits myotube formation as well (Supplemental Fig. S4B–D). Collectively, these findings demonstrate that clenbuterol negatively affects myogenesis, including myotube formation in a cellular model, corroborating the clinical data.

Opposite effects of a β-adrenergic receptor agonist and a β-blocker on ERK phosphorylation in C2C12 cells and human primary myoblasts

Based on the observation that clenbuterol inhibits myogenesis, we aimed to elucidate the specific molecular signaling pathways influenced by β-adrenergic receptor activation. To this end, we investigated the effect of clenbuterol, a β-adrenergic receptor agonist, and atenolol, a β-blocker. Clenbuterol significantly suppressed ERK phosphorylation in both C2C12 cells (Fig. 5A) and human primary myoblasts (Fig. 5B). Notably, this suppression occurred in a PKA-independent manner, suggesting the involvement of alternative downstream signaling pathways in this process. These results suggest that clenbuterol-mediated β-adrenergic activation disrupts ERK activity, potentially contributing to its inhibitory effects on myogenesis, including myotube formation. By contrast, atenolol, a β-adrenergic receptor antagonist, had the opposite effect. Treatment with atenolol increased ERK phosphorylation in both C2C12 cells (Fig. 5C) and human primary myoblasts (Fig. 5D). Within the context of β2-adrenergic signaling, ERK phosphorylation was the only pathway that showed opposing responses to agonist and blocker treatment in both C2C12 cells and human primary myoblasts. In addition, in human primary myoblasts, AKT displayed a regulatory pattern similar to ERK, suggesting inter-species differences in downstream signaling mechanisms (Supplemental Fig. S5). Taken together, these findings demonstrate that β-adrenergic signaling regulates ERK phosphorylation in skeletal muscle cells, with receptor activation and inhibition exerting distinct effects in a context-dependent manner.

Requirement of ERK phosphorylation for myogenic differentiation in muscle cells

Molecular insights into ERK phosphorylation have provided a mechanistic basis for the opposing effects of β-adrenergic agonists and antagonists on myogenesis and myotube formation. To further investigate this, we treated C2C12 cells with LY3214996, a specific inhibitor of ERK signaling involved in C2C12 differentiation and compared its effects with those of clenbuterol on myotube diameter and myogenic differentiation. Treatment with clenbuterol or LY3214996 inhibited myogenic differentiation (Fig. 6A) showing corresponding reductions in myotube diameter (Fig. 6B), MyHC-positive myotube area (Fig. 6C), and fusion index (Fig. 6D). These results confirm the essential role of ERK signaling in myotube formation. Conversely, treatment of C2C12 cells with levobunolol, a β-blocker that increased ERK phosphorylation (Supplemental Fig. S6), promoted myogenic differentiation with a tendency to increase myotube diameter (Fig. 6A, B), reinforcing the positive association between ERK activity and myogenic progression. Collectively, these findings emphasize the pivotal role of ERK phosphorylation in regulating myogenesis and myotube formation, providing a mechanistic explanation for the differential effects of β-adrenergic signaling.

DISCUSSION

This study further explores the relationship between catecholamine levels and muscle mass in a cohort of 830 patients with adrenal incidentalomas, revealing significant associations between catecholamine concentrations and muscle composition. Additionally, muscle cell experiments demonstrated the detrimental effects of β-adrenergic receptor activation on myogenesis and muscle differentiation, providing a mechanistic insight into how catecholamines affect muscle composition and mass regulation.

In line with previous reference studies [22], plasma metanephrine and normetanephrine levels were higher in men than in women in this study cohort, suggesting distinct neuroendocrine profiles between males and females. Building on these overall sex differences, this study further examined the associations between catecholamine levels and muscle mass and quality according to sex. Catecholamine levels were negatively correlated with muscle mass and quality in men but showed weaker correlations in women. Elevated catecholamine levels were associated with reduced NAMA, TAMA, and LAMA. These findings align with previous studies showing that catecholamines, especially when chronically elevated, can contribute to muscle wasting in patients with pheochromocytoma [23], and suggest that even within normal ranges, elevated basal catecholamines can influence muscle composition, emphasizing the importance of understanding sex-specific and physiological differences in catecholamine signaling. This observation is consistent with studies indicating that sympathetic nervous system activity and β-adrenergic responsiveness differ between sexes [14]. The sex-specific effects observed in this study suggest that future therapeutic strategies involving catecholamine modulation should consider these differences to optimize clinical outcomes.

Likewise, catecholamine overproduction, often associated with conditions such as pheochromocytoma or chronic stress, can lead to muscle wasting through several mechanisms. Excessive catecholamines stimulate prolonged β-adrenergic receptor activation, resulting in increased proteolysis and reduced protein synthesis in muscle tissue. This hyperactivation of the adrenergic system also increases cortisol levels, which further exacerbates muscle catabolism. The chronic elevation of catecholamines and cortisol leads to a net loss of muscle protein, contributing to muscle wasting. Additionally, the metabolic shift towards increased gluconeogenesis and reduced glucose uptake in muscle cells deprives muscles of essential nutrients needed for maintenance and growth.

β2-Adrenergic receptor signaling in skeletal muscle involves not only the canonical cAMP–PKA cascade but also alternative pathways such as exchange protein activated by cAMP–mitogen-activated protein kinase (Epac–MAPK), G-protein βγ–phosphoinositide 3-kinase/protein kinase B (Gβγ–PI3K/AKT), and β-arrestin–mediated ERK signaling [3]. In this study, ERK signaling exhibited opposite responses to β-adrenergic agonist and blocker treatment in both C2C12 myoblasts and human primary myoblasts. These findings highlight ERK as an essential regulator of myogenesis, as excessive β2-adrenergic stimulation inhibited ERK and impaired differentiation, while pharmacological inhibition with LY3214996 further suppressed myotube formation, underscoring its critical role in the regulation of proliferation and differentiation [24]. Atenolol, which activates ERK in this study, has also been shown to improve muscle strength and function under conditions where β-adrenergic activation drives muscle degradation, highlighting its therapeutic potential. [25]. Importantly, the ability of β-arrestin–biased β-blockers such as carvedilol to enhance ERK activity and improve muscle contractility suggests that selective ERK modulation may represent a promising therapeutic strategy in catecholamine-induced muscle wasting [26]. A recent metabolomic study in end-stage renal disease (ESRD) patients also demonstrated a significant association between β-adrenergic activation and skeletal muscle wasting, suggesting the potential clinical utility of β-blocker therapy [17]. Consistent with this, our study provides translational evidence supporting ERK-modulating β-blockers as a potential strategy to mitigate muscle loss in ESRD, underscoring the importance of prospective clinical evaluation in muscle wasting disorders.

The comparable pattern of ERK activation observed in human primary myoblasts reinforces the translational relevance of our findings and supports the clinical association between chronic catecholamine excess and reduced skeletal muscle mass. This observation further indicates that the underlying mechanisms involve ERK-dependent pathways, although in vivo validation using additional β-agonists and blockers targeting various β-adrenergic receptor subtypes will be required to fully establish their physiological relevance. Moreover, the finding that AKT signaling exhibited a response similar to ERK in human primary myoblasts indicates that heterogeneous signaling dynamics are likely to exist in human skeletal muscle, emphasizing the importance of accounting for patient-specific mechanisms when developing therapeutic interventions.

This study has several strengths and limitations. The strengths of this study were the large sample size and decade-long data collection, which enhanced the reliability and robustness of the findings. Stratifying the analysis by sex allowed for a detailed understanding of sex-specific correlations between catecholamine levels and body composition. Additionally, AI-based detailed analysis of muscle and fat compartments provided comprehensive insights into the effects of catecholamines. Furthermore, mechanistic experiments using muscle cells demonstrated that catecholamine signaling inhibits muscle cell differentiation, and rescue experiments using β-blockers provided additional support for the current clinical data and previous murine models [25,26]. Moreover, by incorporating key hormonal factors, the study comprehensively addressed physiological pathways linking catecholamine signaling to muscle composition. In addition, by integrating AI-assisted CT quantification with mechanistic in vitro experiments, our study strengthens the biological plausibility of the clinical observations and highlights the value of combining imaging, computational approaches, and molecular biology within a single framework. The limitations of this study include its single-center design, which may restrict generalizability. While the analysis cannot establish causality, the experimental findings help substantiate clinical observations. Moreover, due to the retrospective nature of the cohort, several variables known to affect muscle health could not be collected, including thyroid hormones, smoking status, alcohol consumption, skeletal muscle index, standardized performance measures such as the short physical performance battery. These missing variables represent important limitations and should be incorporated in future prospective studies to improve causal inference and clinical applicability. Taken together, these limitations highlight the need for further research to elucidate the mechanisms underlying these associations and confirm the findings in broader populations, including healthy individuals without adrenal incidentalomas.

In conclusion, this study highlights the complex relationship between catecholamines and muscle mass, suggesting that while catecholamines play a role in muscle growth, chronic or excessive catecholamine exposure may contribute to muscle wasting. β-Adrenergic antagonism emerges as a potential therapeutic strategy for mitigating muscle loss, particularly in conditions associated with elevated catecholamine levels. Furthermore, our findings emphasize the need to consider sex and age-related variations in catecholamine signaling when developing targeted treatments for muscle wasting and sarcopenia. Further research is warranted to validate these findings and explore their implications in diverse populations.

Supplementary Information

Article information

CONFLICTS OF INTEREST

One of the authors is an employee of ClariPi. No potential conflict of interest relevant to this article was reported.

ACKNOWLEDGMENTS

This work was supported and by a grant from the Korea Health Technology R&D Project, through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (RS-2024-00507183). Hyon-Seung Yi was supported by the Basic Science Research Program, through the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT, and Future Planning, Korea (NRF-2021R1A5A8029876 and NRF-2023R1 A2C3006220) and by a grant from the Korea Health Technology R&D Project, through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (RS-2022-KH130308). Ju Yeon Kwak was supported by the National Research Council of Science & Technology (NST) grant by the Korean government (MSIT) (CRC22011-200), and the KRIBB initiative program. We sincerely appreciate the support of Aventi Inc. for providing the MCK::EGFP C2C12 cell line and access to the Incucyte Live-Cell Analysis System, which was essential in this study.

AUTHOR CONTRIBUTIONS

Conception or design: J.L., J.Y.K., J.E.L., H.S.Y. Acquisition, analysis, or interpretation of data: J.L., J.Y.K., H.Y.L., J.S.M., H.J.J., S.K. Drafting the work or revising: J.L., J.Y.K., J.E.L., H.S.Y. Final approval of the manuscript: J.L., J.Y.K., H.Y.L., J.S.M., H.J.J., H.T.N., T.L.N., A.M.A., J.K., S.K., Y.R.Y., J.E.L., H.S.Y.

Fig. 1

Schematics of the abdominal fat and muscle tissues and the tissue analysis algorithm in ClariMetabo. Schematic representation of the abdominal fat and muscle tissues, and the tissue analysis algorithm using ClariMetabo, showing the segmentation and quantification process. CT, computed tomography; MIP, maximum intensity projected; BN, batch normalization; ReLU, rectified linear unit; conv, convolution layer; VFA, visceral fat area; SFA, subcutaneous fat area; HR, Hounsfield range; IMAT, intermuscular adipose tissue; LAMA, low-attenuation muscular area; NAMA, normal-attenuation muscular area; SMA, skeletal muscle area; TAMA, total abdominal muscle area.

Fig. 2

Partial correlation plots showing the correlation between catecholamine levels and age, body mass index, muscle mass, and fat mass by sex. Correlograms were generated using Spearman partial correlation coefficients for metanephrine and normetanephrine to visceral fat area (VFA), subcutaneous fat area (SFA), total fat area (TFA), myosteatosis, intermuscular adipose tissue (IMAT), low-attenuation muscular area (LAMA), normal-attenuation muscular area (NAMA), skeletal muscle area (SMA), total abdominal muscle area (TAMA), 24-hour urinary free cortisol (24h UFC), overnight dexamethasone suppression test (ODST) controlling age, estimated glomerular filtration rate, total protein, blood pressure, and albumin. Data represent single measurements from male (n=415) and female (n=415) patients. (A) Partial correlation plots for male participants. (B) Partial correlation plots for female participants. In the correlogram, the color gradient reflects the direction and magnitude of Spearman partial correlation coefficients, while the size of each circle corresponds to the absolute value of the correlation.

Fig. 3

Representative abdominal computed tomography (CT) scan images of the subjects with high or low catecholamine levels. (A) A male with metanephrine levels of 0.06 nmol/L and normetanephrine levels of 0.79 nmol/L. (B) The abdominal muscle mass from a CT scan of a male with metanephrine levels of 0.77 nmol/L and normetanephrine levels of 2.32 nmol/L. TAMA, total abdominal muscle area; SMA, skeletal muscle area; IMAT, intermuscular adipose tissue; LAMA, low-attenuation muscular area; NAMA, normal-attenuation muscular area.

Fig. 4

β-Adrenergic agonist inhibits myotube formation in muscle creatine kinase (MCK)-enhanced green fluorescent protein (EGFP) C2C12 cells. (A) Schematic representation of the construct used in the MCK::EGFP cell line. (B) Experimental scheme for clenbuterol treatment during C2C12 myogenesis. (C) Representative images of differentiated myotubes treated with clenbuterol during myogenesis. Scale bar, 200 μm. (D) Quantification of green fluorescent protein (GFP) signals in myogenesis by measuring the total green object area using the Incucyte Live-Cell Analysis System (Sartorius). (E) Bar graphs showing the quantification of GFP signals from day 3 to 5. (C, D) Data are shown as mean±standard error of mean (n=12). P values were calculated by the two-way analysis of variance (ANOVA) with Šídák’s multiple comparisons test (D), and with Dunnett’s multiple comparison test (E). DM, differentiation medium; HS, horse serum; CLB, clenbuterol; NS, not significant. ^aP<0.0005; ^bP<0.0001.

Fig. 5

β-Adrenergic receptor agonists suppress extracellular signal-regulated kinase (ERK) phosphorylation, whereas the β-blocker atenolol increases ERK phosphorylation in C2C12 cells and human primary myoblasts. Western blot analysis of ERK phosphorylation signaling following treatment with the β-adrenergic receptor agonist clenbuterol in C2C12 myoblasts for 10 minutes (A) and human primary myoblasts for 30 minutes (B). Western blot analysis of ERK phosphorylation signaling by β-blocker atenolol treatment in C2C12 myoblasts for 10 minutes (C) and human primary myoblasts (D). PKA, protein kinase A; p-PKAc, phospho-protein kinase A catalytic subunit.

Fig. 6

Extracellular signal-regulated kinase (ERK) phosphorylation is required for myogenic differentiation in muscle cells. (A) Representative images of myotubes in differentiated C2C12 cells treated with 1 μM of clenbuterol, atenolol, levobunolol or LY3214996 during myogenic differentiation. Scale bar, 200 μm. (B) Measurement of C2C12 myotube diameters, where larger myotubes appear darker. (C) Fusion index was calculated as the ratio of nuclei within myosin heavy chain (MyHC)-positive myotubes containing ≥3 nuclei to total nuclei. (D) Quantification of green fluorescent protein (GFP) signals in C2C12 myotubes by measuring the total green object area using the Incucyte Live-Cell Analysis System (Sartorius). Data are shown as mean±standard error of mean (n=4) (B, D) and (n=16) (C). P values were calculated by two-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test (B) or an ordinary one-way ANOVA with Dunnett’s multiple comparison test. NS, not significant. ^aP<0.05; ^bP<0.005; ^cP<0.0005; ^dP<0.0001.

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References

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