Divergent alterations in the skeletal muscle and serum proteome in a rodent model of anorexia nervosa and weight recovery

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However, physiological changes during AN and following weight restoration are poorly understood, especially skeletal muscle physiology and how it relates to systemic physiology. In this study, we utilized a rodent model of AN and weight gain to investigate proteomic changes across the skeletal muscle and serum and how these proteomic alterations relate to muscle functional outcomes. Using SOMAscan proteomics, we identified extensive alterations in the skeletal muscle proteome during 30 days of simulated AN, with the majority of these changes persisting after weight restoration. Muscle mass and strength were not fully recovered after weight restoration, aligning with sustained dysregulation of proteins related to apoptosis, calcium handling, and satellite cell proliferation in skeletal muscle. By contrast, serum proteomic changes were modest during AN but markedly altered and largely distinct from muscle during recovery. TRAIL (TNF-related apoptosis-inducing ligand) emerged as a potential non-invasive biomarker of muscle health as TRAIL content correlated between muscle and serum as well as muscle functional assessments. Proteomic biomarkers of muscle versus systemic AN are largely unique, underlining the complexity of this illness and the need for novel analytical approaches; however, TRAIL dysregulation may be a conserved feature across tissues and should be further investigated as potential non-invasive biomarker of muscle health and a possible mechanistic target for AN-induced muscle loss. Health sciences/Diseases/Psychiatric disorders Health sciences/Biomarkers/Predictive markers Biological sciences/Physiology Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Anorexia Nervosa (AN) and its associated complications remain a significant public health challenge for clinicians, patients, and their families. Anorexia Nervosa affects ~ 4% of women and 0.5% of men in the United States 1 – 3 , and these percentages correspond to millions of individuals and billons of dollars in healthcare expenses 4 , 5 . AN is also one of the most deadly psychiatric disorders; individuals with AN have a 4.5-fold increased mortality risk compared to those without AN 6 . Despite its prevalence and lethality, treatment options for those with AN are limited and often ineffective, resulting in prolonged disease. This sustained disease resistance necessitates novel research avenues to understand the complex interplay of physiology and psychology in this population. Along with fat loss, the implications of which largely through leptin have been studied extensively 7 , 8 , there is a concurrent loss of skeletal muscle during AN. This skeletal muscle loss has been largely uninvestigated across clinical or basic studies of AN. However, recent evidence demonstrates skeletal muscle loss is a significant predictor of morbidity across multiple pathologies such as cancer, aging, others 9 . It is therefore plausible that low muscle mass during AN may be an under investigated contributor to the overall pathophysiology of the condition and contribute to treatment challenges. Our group’s recent systematic review found ~ 25% lower skeletal muscle mass/strength in those with AN compared to healthy controls, which is not fully recovered after weight restoration (~ 9% lower) 10 . Other recent studies have provided some mechanistic insights for low muscle mass in those with AN. For example, our previous works found altered Leukemia Inhibitory Factor Receptor (LIF-R) in the serum of individuals with AN 11 . LIF-R is a key modulator of cancer-associated muscle wasting 12 . LIF-R is also one of the few proteins that differentiated individuals with AN from healthy controls. Other recent works suggest acute alterations to both protein synthetic signaling during AN, which are only partially restored with weight recovery 13 . Emerging evidence therefore suggests that skeletal muscle physiology may be an important contributor to physiological alterations concurrent with AN. Indeed, the myostatin-activin-follistatin pathway has recently been implicated as a key moderator of loss of muscle functional performance in relative energy deficiency in sport (RED-S) 14 . Yet, to our knowledge, in-depth biological evaluation of the skeletal muscle during extreme caloric deprivation characteristic of AN does not exist. Currently, most of our biological understanding of physiological alterations during AN are based solely on blood-based biomarkers. While these markers are clearly important and less invasive than alternative approaches, more direct assessment of skeletal muscle alterations are necessary to fully understand the biological implications of skeletal muscle health during AN and following weight restoration. In this study, we leveraged our recently published rodent model of AN and recovery to evaluate proteomic alterations within the skeletal muscle and the serum during AN and following weight restoration. We find broad proteomic alterations to the skeletal muscle that are not resolved following weight restoration. Moreover, we find proteomic changes in the skeletal muscle are unique in the serum, but that conserved biomarkers of systemic and skeletal muscle disruption during AN may exist. Taken together, these data strongly imply physiological and musculoskeletal consequences of AN are longer lasting and more difficult to identify than previously anticipated. Methods Animals Experiments : All procedures were approved by the Beth Israel Deaconess Medical Center Institutional Animal Care and Use Committee (AUP: 009-2022). We used our recently refined model of AN and AN recovery, which includes group housing rodents for the duration of simulated AN instead of single housing 15 . These rodents are subsets of previously reported studies from our laboratory 13 . All rats were housed in a temperature and humidity-controlled facility kept at ~ 23 o C with a 12:12 hour light:dark cycle. At 8 weeks of age, female Sprague Dawley rats began simulated AN protocol, consisting of ~ 50–60% food restriction. Food consumption was assessed 3 days prior to initiation of simulated AN to obtain a baseline food measurement. AN rats were fed ~ 8:00 AM each day, wherein they were moved to individual cages for feeding and allowed up to 2 hours to consume food. Rats were then returned to group housing until feeding the following day. Simulated AN occurred for 30 days, afterwards one cohort of rats (AN, n = 8) were anesthetized with 2.5% isoflurane and gastrocnemius muscles collected and snap frozen in liquid nitrogen for further analysis. Rats were euthanized while under anesthesia by cardiac puncture. Another cohort of rats was provided food ad libitum after simulated AN to mirror weight recovery (AN-R, n = 8). AN-R rats continued recovery interventions for 30 days. Afterwards, rats were anesthetized with isoflurane (~ 3%), and gastrocnemius muscles collected for further analysis. Control (CON) rats consumed food ad libitum throughout the protocol and were age-matched to AN-R. All gastrocnemius muscles and serum were collected in the fed state (~ 90 min after feeding) using a standardized amount of food for all groups (~ 3 grams of food). Nerve stimulated force production Maximal force production of the plantar flexor muscles (encompassing the gastrocnemius) was completed ~ 24 hours prior to euthanasia and completed similar to our previous reports 16 . Briefly, rats were anesthetized with ~ 2.5% isoflurane. Their tibial nerve was then stimulated with electrode needles to plantar flexor muscle contraction. Small twitches (10 Hz) were used to ensure appropriate needle placement; thereafter rats received one maximal stimulation at 200 Hz for 200 ms. Graphs of maximal tetanus were visually inspected to ensure a full tetanic contraction was achieved. Grip Strength Maximal rear paw grip strength was completed ~ 24 hours prior to euthanasia as we have described 17 . Briefly, rats were gently restrained by a trained investigator and the two hindlimb paws placed on a grip bar attached to a force transducer. The rat was then gently pulled from the transducer until the rat released both paws. The corresponding force was then recorded. The procedure was repeated three times and the maximal measurement was used for analysis. SomaScan Serum and skeletal muscle tissue underwent SOMAscan discovery proteomics at the Genomics, Proteomics, Bioinformatics and Systems Biology Center at Beth Israel Deaconess Medical Center. Briefly, the SOMAscan platform is aptamer-based technology that utilizes single-stranded DNA chemically modified to enhance protein binding with high specificity to measure > 1,300 proteins 18 , 19 . Samples were prepared following manufacturer’s recommended protocols. Data quality control and normalization procedures were followed as per previous publications 20 , 21 . Statistical and Pathway Analysis : Phenotypic data were analyzed with SAS (SAS® Studio Release: 3.81, SAS Institute Inc. Cary, NC) with a one-way ANOVA and a Tukey-adjusted post-hoc p < 0.05. Differentially expressed proteins were determined using SomaLogic’s DataDelve™ Statistics analytical platform. Three AN samples were found to have significantly divergent proteomic profiles compared to the remainder of the group. Upon further examination of the data, we noted these animals did not lose as much muscle strength or bodyweight compared to other rats within the group. We therefore set those rats aside for subsequent proteomic analysis. Similarly, three CON serum profiles were set aside as outliers due to substantial differences in their proteomic signatures compared to the rest of the group. We analyzed up and down-regulated proteins among three specific comparisons: AN v. CON, AN v. AN-R and CON v. AN-R. To refine which processes are persistently dysregulated, we performed pathway analyses on proteins persistently dysregulated between AN and AN-R with background correction (background: all proteins detected in dataset). No pathways reached significance at adjusted p < 0.05, so we used a non-adjusted p < 0.001 as our significance threshold. After differentially altered proteins were assessed, we used ShinyGO 0.82, an online web browser platform, for enrichment analyses to evaluate altered Gene Ontology (GO) pathways 22 , 23 . Correlations were assessed with GraphPad Prism Version 10.2.3. All raw and analyzed data for this project can be found on this project’s Open Science Framework Page ( https://osf.io/vhy8f/overview?view_only=e721e9b44fa0499195e4cab54c6615d3 ) Results Simulated AN resulted in lower muscle size and strength, which were not fully resolved following weight gain Simulated AN resulted in significant differences in bodyweight compared to CON (~ 36%, p < 0.001, Table 1 ); however, with refeeding bodyweight differences were no longer present between AN-R and CON (~ 5%, p = 0.066, Table 1 ). AN resulted in lower rear paw grip strength (~ 40%) which was not restored in AN-R rats compared to CON (~ 22% lower, p = 0.010, Table 1 ). Maximal plantar flexion was ~ 25% lower in AN rats compared to CON (p < 0.001, Table 1 ). With weight recovery, plantar flexion in AN-R was not different from CON (~ 9% lower, p = 0.105, Table 1 ). Gastrocnemius mass was ~ 32% lower in AN compared to CON (p < 0.001) and with weight recovery, AN-R was still ~ 12% lower compared to CON (p = 0.003, Table 1 ). Table 1 CON AN AN-R Bodyweight (g) 234.13 ± 4.79 a 148.80 ± 1.70 b 221.18 ± 4.28 a Rear Paw Grip (N) 8.44 ± 0.22 a 5.12 ± 0.43 b 7.0167 ± 0.23 c Maximal Plantar Flexion (mN) 120.54 ± 2.30 a 90.35 ± 4.79 b 109.87 ± 2.97 a Gastrocnemius Mass (g) 1.170 ± 0.0247 a 0.7919 ± 0.0123 b 1.0319 ± 0.0361 c Phenotypic data of bodyweight and muscle outcome variables. Data are depicted as Mean + SEM. Different letters represent statistically different at Tukey adjusted p < 0.05. CON = control, AN = anorexia nervosa, AN-R = anorexia nervosa + recovery. N = 8/group Simulated AN resulted in large changes to the skeletal muscle proteome, which were not restored following weight gain We sought to examine alterations to the muscle proteome as a potential driver of muscle mass and functional losses during food-restricted anorexia nervosa (AN) and subsequent recovery with re-feeding (AN-R) in the gastrocnemius muscle. With AN compared to CON timepoint, there were 112 upregulated and 256 downregulated differentially abundant proteins (Fig. 1 A). The top 3 differentially abundant upregulated proteins were PFKM, RBM3 and KCTD13 (Fig. 1 B). The top 3 abundant downregulated proteins were DHP5, FGF8 and ARL14 (Fig. 1 C). The remaining top 10 proteins in each direction are shown in Figs. 1 B and 1 C. There were only 5 proteins that were altered with AN that returned to baseline with recovery (Fig. 1 D), indicating that many of the alterations that occurred during AN were not resolved with weight gain. Instead, we see persistent alteration of the proteome after the recovery phase (AN-R), with 245 upregulated and 377 downregulated proteins compared to CON. We compared the overlap in differentially expressed proteins between the AN and AN-R proteomes and found 65 of 112 (58%) proteins upregulated with AN remained upregulated at the AN-R timepoint relative to CON, and 201 of 256 proteins (78%) downregulated with AN remained downregulated at AN-R timepoint (Fig. 1 E and 1 F). Some of the top upregulated pathways included protein-DNA complex assembly, apoptotic signaling related to p53, and glycoside metabolic processing (Fig. 1 E). Downregulated pathways related to cation transport, muscle satellite cell proliferation, calcium binding, synapse organization and others (Fig. 1 F). Persistent repression of proteins related to the synapse, and binding/transport of cations which are essential for muscle contraction could perhaps explain why volitional muscle strength was not recovered in AN-R rats compared to CON. Simulated AN resulted in changes to serum proteome; however, AN-R was more altered compared to AN We next wanted to determine if the serum proteome reflects the muscle proteome during AN and AN-R to identify potential biomarkers of muscle function and size that could be obtained from blood, as muscle biopsies are quite invasive. Only 15 proteins were differentially expressed after AN compared to CON (2 up and 13 down, Fig. 2 A). No proteins were up or downregulated in muscle and serum simultaneously at any time point (Supplement A and B). Only one protein, GLTP, a glycolipid transfer protein, was upregulated in muscle while downregulated in serum. Most changes in the serum proteome occurred with refeeding, with 317 and 102 upregulated and downregulated proteins, respectively, in AN-R versus AN (Fig. 2 A, 2 C, 2 D). We also performed pathway analyses on those proteins (p < 0.001). Upregulated GO terms relate to calcium ion binding and TRAIL (TNF-related apoptosis-induced ligand)-activated apoptotic signaling, apoptotic signaling (Fig. 2 F). Downregulated GO terms related to telomere maintenance, Cajal body localization, and endopeptidase activity and others including skeletal muscle tissue regeneration (Fig. 2 F) Several proteins are correlated between the skeletal muscle and serum across different experimental timepoints While general patterns between the muscle and serum proteome were not present, we next sought to evaluate if any individual proteins showed convergent patterns between muscle and serum samples. We used animal-matched normalized proteomic values for each sample across timepoints to calculate correlations between muscle and serum. Once significant correlations were identified, individual proteins were further evaluated to assess if correlations were being driven by outliers. We found six proteins that had a significant correlation between muscle and serum (Fig. 3 A-F). Of those six, NDK7 had the strongest correlation between muscle and serum (r = 0.847, p = 0.0017, Fig. 3 A), followed by NAB2 (r = 0.797, p = 0.0007, Fig. 3 B), and TRAIL (TNFSF10, r = 0.642, p = 0.0014, Fig. 3 C). Of proteins that correlate between biological samples, several also correlated with muscle function We conducted correlations on a few key proteins in muscle and serum in relation to functional outcomes. MSRB1 (methionine sulfoxide reductase B1) was one of the few proteins that was altered with AN and restored in AN-R. Moreover, previous work suggests selenium metabolism is important moderator for disease severity during AN 24 and may be a key driver of muscle health 25 . Therefore, we first correlated muscle MSRB1 to key metrics of muscle health and performance. Overall MSRB1 had strong correlations with maximal plantar flexion (r = 0.7965, p < 0.0001), rear paw grip strength (r = 0.7273, p = 0.002), muscle fiber cross-sectional area (r = 0.5902, p = 0.0048), and gastrocnemius mass (r = 0.7441, p = 0.0001, Fig. 4 A- 4 D), overall implying some of the proteomic signatures from our rats corresponded to functional outcomes. Given the previous correlations between serum and muscle proteins for NDK7 and NAB2, we next evaluated if the serum content of these proteins correlated to functional outcomes. Overall, neither protein strongly correlated with any functional parameters (Supplementary Fig. 2), suggesting serum content of these proteins does not appear related to muscle function. However, TRAIL serum content did correlate to muscle functional outcomes including maximal plantar flexion (r = 0.7308, p = 0.0006), rear paw grip strength (r = 0.8371, p < 0.0001), muscle fiber cross-sectional area (r = 0.6876, p = 0.0016), and gastrocnemius mass (r = 0.8333, p < 0.0001, Fig. 4 E- 4 H). Together, TRAIL was the only protein that correlated between muscle and serum and correlated with functional parameters of muscle strength and size. Discussion In this study we utilized a novel proteomic technique to evaluate the proteomic signature of both skeletal muscle and serum in a rodent model of AN and subsequent weight recovery. We found significant alterations to the skeletal muscle proteome during AN that are not resolved with weight recovery. These broad proteomic perturbations mirror phenotypic alterations within the skeletal muscle, with sustained alterations to muscle function despite weight gain. However, within the serum of these same animals, we find relatively minor proteomic changes in AN rats and instead find dramatic alterations in the proteomic signature of the AN-R rats compared to healthy controls. Moreover, the broad proteomic changes within the serum are not generally reflected in the skeletal muscle proteomic changes. In aggregate, these data demonstrate the profound alterations to physiology during AN and suggest prolonged physiological alterations due to AN. One of the initial goals of this study was to evaluate overlap of serum and skeletal muscle proteomic signatures in our model, potentially allowing for a less invasive and faster (compared to maximal muscle testing) method to evaluate skeletal muscle health. Broadly, we find the proteomic signatures of serum and muscle did not overlap. Specifically, within skeletal muscle we note large changes to the proteome during AN, many of which are not resolved with weight recovery. Contrastingly, within the serum, we find relatively modest changes in the proteome during AN compared to CON, yet with weight recovery (AN-R) there were large alterations compared to either AN or CON. There are a few reasons the skeletal muscle proteome may not fully reflect circulating proteome. First, the serum proteome reflects paracrine activity of the skeletal muscle, but also of all other organs within the body. Given that AN is a whole-body pathology, it is likely other tissues (liver, fat, etc.) are also experiencing profound proteomic modifications. The combination of all of these tissue-specific proteomic changes and corresponding alterations to paracrine activity of these tissues would result in systemic proteomic changes that do not directly mirror tissue-specific proteomic changes. Additionally, the skeletal muscle proteomic changes may reflect alterations to tissue specific biology or changes to the paracrine activity of the skeletal muscle, at present we cannot directly evaluate which transformations within the skeletal muscle tissue are directly altering myokine release from the skeletal muscle and this warrants further investigation. Regardless, our findings suggest proteomic alterations to skeletal muscle and blood are unique during AN and following weight restoration, implying more nuanced or novel techniques will likely be necessary to fully understand the biological ramifications of AN and to develop possible future biomarkers of muscle health that do not require an invasive muscle biopsy or maximal muscle strength assessment. Our data seemingly contrasts other proteomic studies evaluating the serum proteomic alterations during the low weight phase of AN. These differences may in part be due to controlling for multiple comparisons with the SomaScan platform that evaluates significantly more proteins compared to other proteomic platforms as well as the low number of animals per group. However, prior studies in individuals with AN have tended to suggest inflammatory/cytokine signaling and complement may be key pathways in the etiology of AN 11, 26–28 . Moreover, a recent study evaluating proteomic alterations during acute starvation found similar signaling pathways such as inflammation and complement signaling 29 , suggesting this may be an important physiological alteration that is conserved during severe energy restriction regardless of psychological status. Within our data sets, we also do note some signaling changes that may also support this mechanism such as TRAIL cytokine signaling within the serum and muscle (discussed further below) as well as IL-1Beta and T-helper cell differentiation within the skeletal muscle. However, our data strongly suggests, broad proteomic changes within the skeletal muscle are not reflected in the serum either during acute starvation or during weight restoration. These findings provide context for previous studies and future studies on the reliability of correlating serum findings with skeletal muscle health and further highlight the need for new technologies to non-invasively assess muscle health. Within the skeletal muscle, many of the top up-regulated and down-regulated proteins during AN are relatively novel in the skeletal muscle literature. These new discoveries may open new avenues to evaluate the relative roles of these proteins in skeletal muscle biology and how they affect systemic health. For example, PFKM is known to be the muscle-specific isoform of PFK, a key glycolytic enzyme. Given limited glucose availability is inherent to AN, the greater abundance of PFKM is a bit paradoxical; however, this specific isoform of PFK may have hitherto unknow roles in muscle biology. Additionally, KCTD13 has previously been reported as a possible genetic contributor to the development of neuropsychiatric diseases 30 . Genetic deletion of KCTD13 inhibited DNA synthesis and proliferation in human-induced pluripotent stem cells 30 . RBM3 is the only top differentially expressed proteins that has previously been reported in skeletal muscle literature. Specifically, the protein is known to be induced in cold environments such as hibernating mammals 31 . More recently, mechanistic studies of RBM3 have demonstrated it is sufficient to improve mitochondrial metabolism and myoblast differentiation 32 . Rodent studies have found RBM3 is elevated during physiological caloric restriction during aging, which is thought to contribute to caloric restriction’s longevity effects 33 . Since there was greater RBM3 content in the present study, it is difficult to fully interpret its role in skeletal muscle physiology and the nuances of RBM3 in relation to pathological (ie starvation/AN) vs. physiological caloric restriction is warranted. DHPS, FGF8, and ARL14 were the top differentially abundant down-regulated proteins in AN skeletal muscle compared to CON. DHPS is a key protein necessary for activation of eIF5A, which appears necessary for satellite cell activation and presumably muscle growth 34 – 36 . Moreover, loss of DHPS in zebrafish resulted in significant defects in pancreatic growth, accompanied by changes in gene expression related to mRNA translation, neurogenesis, and stress pathways 37 . Similarly, FGF8 is known to facilitate myogenesis and prevent fat accumulation within skeletal muscle 38 . ARL14 has only been reported within the context of cancer 39 , so its role in skeletal muscle biology remains unclear. In aggregate, considering the top down-regulated proteins are related to muscle growth and remain depressed during AN-R, these results strongly suggest muscle growth pathways are severely altered during AN and simple weight recovery (with duration matching length of starvation) is not sufficient to restore these pathways. Broadly, the overall patterns in the proteome of the skeletal muscle compared to serum did not have significant overlap, suggesting that broadly serum protein markers are likely not viable methods to estimate muscle health in humans. However, three proteins broadly have values that correlated within the serum and the skeletal muscle, NDK7, NAB2, and TRAIL. While NDK7 and NAB2 overall had strong correlations within the serum and skeletal muscle (r = 0.847 and r = 0.798 respectively), they did not generally correlate with either muscle mass or muscle function outcomes (r range = 0.328–0.541). Suggesting neither of those proteins are necessarily indicative of muscle health per se. TRAIL content in the skeletal muscle and serum correlated (r = 0.641) and TRAIL content within the serum correlated strongly with muscle size and functional outcomes (range = 0.688–0.837). Functionally, TRAIL is a cytokine within the TNF family of cytokines 40 . Within skeletal muscle, the exact role for TRAIL within skeletal muscle health is not fully clear, with some studies finding TRAIL to negatively regulate muscle differentiation 41 , whereas others have indicated TRAIL is an important moderator of muscle differentiation and Akt signaling 42 . Given the known phenotypic alterations to muscle health in this particular model, the latter explanation of TRAIL activity seems likely; however, more mechanistic studies are likely necessary to fully understand the relevance of TRAIL to muscle biology and subsequent health. Regardless, these correlational data suggest serum TRAIL content may be a viable candidate for estimating muscle health during AN and subsequent recovery in human patients. Previous works in patients with AN have also found lower serum TRAIL content 11 , 28 , overall providing additional clinical robustness to our current findings. Therefore, TRAIL content may be a viable candidate to non-invasively estimate muscle health in patients with AN or recovering from AN and additional studies to directly assess this hypothesis in humans are warranted. There are a few limitations of this study that should be acknowledged. We are likely not fully recapitulating the nuances of a complex psychological disorder such as AN with our rodent model. However, as some of our results in rats appear to replicate some results found in humans, our rodent model appears to at least mimic the physiological responses to severe caloric restriction inherent to AN. Additionally, for this study we utilized a different proteomics technology (SomaScan) compared to other previously reported proteomic based analysis (O-link or GC-MS based platforms). These differences between methods can make direct comparisons between studies difficult. For example, due to the broad range of the SomaScan platform (> 7000 proteins identified) and the mathematical need to correct for multiple comparisons, our analysis may have omitted some proteins that other analysis with smaller protein identification ranges may have identified as significant. Moreover, we cannot account for possible differences in the serum or skeletal muscle proteome based on age. Our CON and AN-R rats are four weeks older than our AN rats in order to age-match these two groups. Therefore, it is possible that some differences may be due to maturation of the rats during the course of the study. Similarly, our results reflect a duration of recovery matching the duration of simulated AN. We cannot determine if proteomic changes are persistent in perpetuity or if these proteomic changes would be resolved given additional recovery time. Finally, a proteomics analysis of the selected tissues only reflects the protein content within that specific tissue and does not indicate what cell types these proteins are being synthesized from (multiple tissues can contribute to the proteomic signature in the serum and there are multiple different cell types within skeletal muscle). These limitations can be addressed in future studies using more targeted proteomic or transcriptomic approaches, such as single-fiber approaches or single-cell RNA sequencing. In this study we evaluated the proteome of skeletal muscle and serum from a rodent model of AN and subsequent weight recovery. We find the skeletal muscle proteome is largely altered during AN and remains altered during weight restoration. Moreover, the serum proteome is largely altered during weight restoration. These results appear to suggest physiological responses to AN and weight recovery are longer-lasting than previously anticipated. Moreover, we find changes to the skeletal muscle and serum proteome do not broadly overlap, with a few exceptions. Specifically, TRAIL content appears to correlate across tissues and functional outcomes, suggesting assessment of this protein may serve as a viable biomarker in the future and/or may be a possible physiological moderator of muscle derangements during AN. Future research should seek to further clarify the relationship between TRAIL (circulating and muscle specific) and muscle functional outcomes in human patients with AN. If the results from our current investigation are consistent in humans, TRAIL content may offer a novel tool to evaluate physiological recovery from AN that is independent of psychological assessments. Declarations Conflict of Interest: The authors declare no conflicts of interests related to this work. Data Availability Statement: Raw data and associated statistical code are available at our Open Science Framework page for this project at: https://osf.io/vhy8f/overview?view_only=e721e9b44fa0499195e4cab54c6615d3 Acknowledgements : This study was supported by the Beth Israel Deaconess Medical Center Translational Spark Hubs Grant, (M.E.R/C.M./L.B.), NIH grant K12HD051959 (M.E.R.) and the Arkansas Integrative Metabolic Research Center Pilot Grant (M.E.R.) as part of the AIMRC Center for Biomedical Research Excellence (COBRE) grant (5P20GM139768). We would like to thank Eliza Garland, Sheridyn Pinkham, Madisyn Hancock, and Sophie Jalkut for their assistance with this project. References Smink FR, van Hoeken D, Hoek HW. Epidemiology, course, and outcome of eating disorders. Curr Opin Psychiatry 2013; 26 (6) : 543-548. AE vE, D vH, HW H. Incidence, prevalence and mortality of anorexia nervosa and bulimia nervosa. Current opinion in psychiatry 2021; 34 (6). Micali N, Martini MG, Thomas JJ, Eddy KT, Kothari R, Russell E et al. Lifetime and 12-month prevalence of eating disorders amongst women in mid-life: a population-based study of diagnoses and risk factors. BMC Med 2017; 15 . Mitchell JE, Myers T, Crosby R, O’Neill G, Carlisle J, Gerlach S. Health Care Costs in Patients with Eating Disorders. Int J Eat Disord 2009; 42 (6) : 571-574. de Oliveira C, Colton P, Cheng J, Olmsted M, Kurdyak P. The direct health care costs of eating disorders among hospitalized patients: A population-based study. Int J Eat Disord 2017; 50 (12) : 1385-1393. Søeby M, Gribsholt SB, Clausen L, Richelsen B. Overall and cause-specific mortality in anorexia nervosa; impact of psychiatric comorbidity and sex in a 40-year follow-up study. The International journal of eating disorders 2024; 57 (9). Khan SM, Hamnvik OP, Brinkoetter M, Mantzoros CS. Leptin as a modulator of neuroendocrine function in humans. Yonsei medical journal 2012; 53 (4). Chrysafi P, Perakakis N, Farr OM, et al . Leptin alters energy intake and fat mass but not energy expenditure in lean subjects. Nature communications 2020; 11 (1). Zhou HH, Liao Y, Peng Z, Liu F, Wang Q, Yang W. Association of muscle wasting with mortality risk among adults: A systematic review and meta-analysis of prospective studies. Journal of cachexia, sarcopenia and muscle 2023; 14 (4). Rosa-Caldwell ME, Eddy KT, Rutkove SB, Breithaupt L. Anorexia nervosa and muscle health: A systematic review of our current understanding and future recommendations for study. The International journal of eating disorders 2022. Breithaupt L, Holsen LM, Ji C, et al . Identification of State Markers in Anorexia Nervosa: Replication and Extension of Inflammation-Associated Biomarkers Using Multiplex Profiling. Biological psychiatry global open science 2024; 4 (5). Kandarian SC, Nosacka RL, Delitto AE, et al. Tumour-derived leukaemia inhibitory factor is a major driver of cancer cachexia and morbidity in C26 tumour-bearing mice. Journal of cachexia, sarcopenia and muscle 2018; 9 (6). Rosa-Caldwell M, Breithaupt L, Kaiser U, Muhyudin R, Rutkove S. Changes in muscle strength and moderators of protein turnover in a rodent model of anorexia nervosa and recovery. Journal of Nutritional Physiology 2025; 4 : 100010. Angelidi AM, Stefanakis K, Chou SH, et al. Relative Energy Deficiency in Sport (REDs): Endocrine Manifestations, Pathophysiology and Treatments. Endocrine reviews 2024; 45 (5). Rosa-Caldwell, E. M, Breithaupt, L., Kaiser, B. U et al. A refined rodent model of anorexia nervosa: Simulating state-specific effects of caloric restriction and weight restoration. Physiological Reports 2024; 12: e16092. Rosa-Caldwell ME, Mortreux M, Wadhwa A, et al. Sex differences in muscle health in simulated micro- and partial-gravity environments in rats. Sports Med Health Sci. 2023; 5 (4):319-328. Rosa-Caldwell ME, Mortreux M, Wadhwa A, et al. Influence of gonadectomy on muscle health in micro- and partial-gravity environments in rats. J Appl Physiol (1985). 2023; 134 (6):1438-1449. Gold L, Ayers D, Bertino J et al. Aptamer-based multiplexed proteomic technology for biomarker discovery. PloS one 2010; 5 (12). Rohloff JC, Gelinas AD, Jarvis TC, et al. Nucleic Acid Ligands With Protein-like Side Chains: Modified Aptamers and Their Use as Diagnostic and Therapeutic Agents. Molecular therapy Nucleic acids 2014; 3 (10). Candia J, Cheung F, Kotliarov Y, et al. Assessment of Variability in the SOMAscan Assay. Scientific reports 2017; 7 (1). Candia J, Daya GN, Tanaka T, Ferrucci L, Walker KA. Assessment of variability in the plasma 7k SomaScan proteomics assay. Scientific reports 2022; 12 (1). Ge SX, Jung D, Yao R. ShinyGO: a graphical gene-set enrichment tool for animals and plants. Bioinformatics (Oxford, England) 2020; 36 (8). Gene Ontology Consortium, Aleksander SA, Balhoff J, et al. The Gene Ontology knowledgebase in 2023. Genetics 2023; 224 (1). Strumila R, Lengvenyte A, Olie E, et al. Selenium deficiency is associated with disease severity, disrupted reward processing, and increased suicide risk in patients with Anorexia Nervosa. Psychoneuroendocrinology 2022; 140 . Wang Q, Huang J, Zhu K, Zuo W. Selenium and Skeletal Muscle Health in Sports Nutrition. Nutrients 2025; 17 (11). Samodova D, Hoel A, Hansen TH, et al. Plasma proteome profiling reveals metabolic and immunologic differences between Anorexia Nervosa subtypes. Metabolism: clinical and experimental 2024; 152 . Adams DM, Reay WR, Cairns MJ. Multiomic prioritisation of risk genes for anorexia nervosa. Psychological medicine 2023; 53 (14). Nilsson IAK, Millischer V, Göteson A, et al. Aberrant inflammatory profile in acute but not recovered anorexia nervosa. Brain, behavior, and immunity 2020; 88 . Commissati S, Cagigas ML, Masedunskas A, et al. Prolonged fasting promotes systemic inflammation and platelet activation in humans: A medically supervised, water-only fasting and refeeding study. Molecular metabolism 2025; 96 . Kizner V, Naujock M, Fischer S, et al. CRISPR/Cas9-mediated Knockout of the Neuropsychiatric Risk Gene KCTD13 Causes Developmental Deficits in Human Cortical Neurons Derived from Induced Pluripotent Stem Cells. Molecular neurobiology 2020; 57 (2). Zhu X, Bührer C, Wellmann S. Cold-inducible proteins CIRP and RBM3, a unique couple with activities far beyond the cold. Cellular and molecular life sciences : CMLS 2016; 73 (20). Dey P, Rajalaxmi S, Saha P, et al. Cold-shock proteome of myoblasts reveals role of RBM3 in promotion of mitochondrial metabolism and myoblast differentiation. Communications biology 2024; 7 (1). Hettinger ZR, Confides AL, Vanderklish PW, Sidhom S, Masternak MM, Dupont-Versteegden EE. Skeletal muscle RBM3 expression is associated with extended lifespan in Ames Dwarf and calorie restricted mice. Experimental gerontology 2021; 146 . Conjard-Duplany A, Osseni A, Lamboux A, et al. Muscle mTOR controls iron homeostasis and ferritinophagy via NRF2, HIFs and AKT/PKB signaling pathways. Cellular and molecular life sciences : CMLS 2025; 82 (1). Zhang Q, Han W, Wu R, et al. Spermidine-eIF5A axis is essential for muscle stem cell activation via translational control. Cell discovery 2024; 10 (1). Kar RK, Hanner AS, Starost MF, et al. Neuron-specific ablation of eIF5A or deoxyhypusine synthase leads to impairments in growth, viability, neurodevelopment, and cognitive functions in mice. The Journal of biological chemistry 2021; 297 (5). Anderson CM, Kulkarni A, Maier B, et al. Hypusinated and unhypusinated isoforms of the translation factor eIF5A exert distinct effects in models of pancreas development and function. The Journal of biological chemistry 2025; 301 (2). Otsuka T, Kan HM, Mengsteab PY, Tyson B, Laurencin CT. Fibroblast growth factor 8b (FGF-8b) enhances myogenesis and inhibits adipogenesis in rotator cuff muscle cell populations in vitro. Proceedings of the National Academy of Sciences of the United States of America 2024; 121 (1). Zhang B, Xu A, Wu D, et al. ARL14 as a Prognostic Biomarker in Non-Small Cell Lung Cancer. Journal of inflammation research 2021; 14 . Wiley SR, Schooley K, Smolak PJ et al. Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity 1995; 3 (6). Kim D, Singh N, Waldemer-Streyer RJ, Yoon MS, Chen J. Muscle-derived TRAIL negatively regulates myogenic differentiation. Experimental cell research 2020; 394 (1). Toffoli B, Tonon F, Tisato V, et al. TRAIL/DR5 pathway promotes AKT phosphorylation, skeletal muscle differentiation, and glucose uptake. Cell death & disease 2021; 12 (12). Additional Declarations The authors have declared there is NO conflict of interest to disclose Supplementary Files SupplementFig1.jpg Supplemental Figure 1 Legend. Correlation between muscle NDK7 protein expression and A) maximal plantarflexor strength. B) rear paw grip, C) gastrocnemius CSA, D) gastrocnemius mass; Correlation between serum NAB2 protein expression and E) maximal plantarflexor strength, F) rear paw grip, G) gastrocnemius CSA, H) gastrocnemius mass SupplementFig2.jpg Supplemental Figure 2 Legend. A) Venn diagram comparing up and downregulated proteins between AN vs CON in muscle and serum. B) Venn diagram comparing up and downregulated proteins between AN-R vs AN in muscle and serum. C) Venn diagrams comparing number and % of differentially abundant upregulated proteins in serum between AN-R vs CON and AN-R vs AN. D) Venn diagrams comparing number and % of differentially abundant upregulated proteins in serum between AN-R vs CON and AN-R vs AN. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8079717","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":544050076,"identity":"9d69c8e9-23a2-44e4-9024-4fea62ba823e","order_by":0,"name":"Megan 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1","display":"","copyAsset":false,"role":"figure","size":261806,"visible":true,"origin":"","legend":"\u003cp\u003eA) Number of differentially abundant proteins between conditions in muscle. Blue font: upregulated. Red front: downregulated. CON: control. AN: Anorexia Nervosa Timepoint. AN-R: Anorexia Nervosa- Recovery Timepoint. B) Top 10 upregulated proteins in AN vs CON. C) Top 10 Downregulated in AN vs CON. D) Fold-changes of the differentially abundant proteins between AN-R and AN through experimental time-course (2 up, 3 down, note: also differentially abundant from AN vs CON). E) Venn diagram comparing number and % of proteins upregulated between AN vs CON and AN-R vs CON. F) Venn diagram comparing number and % proteins downregulated between AN vs CON and AN-R vs CON. All data presented in A-D are adj. p\u0026lt;0.05. E and F are p\u0026lt;0.001. Data presented as mean ± SEM.\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8079717/v1/c803e2e78ff6e8427a67fd82.jpg"},{"id":96157761,"identity":"554f6a71-bd7d-4681-9f8a-860cfed07b0e","added_by":"auto","created_at":"2025-11-18 08:36:59","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":224110,"visible":true,"origin":"","legend":"\u003cp\u003eA) Number of differentially abundant proteins between conditions in serum. Blue font: upregulated. Red front: downregulated. CON: control. AN: Anorexia Nervosa Timepoint. AN-R: Anorexia Nervosa- Recovery Timepoint. B) Differentially abundant proteins in AN vs CON. C) Top 10 Downregulated in AN-R vs AN. D) Top 10 proteins downregulated in AN-R vs AN. E) Enriched GO terms from proteins upregulated with AN-R vs AN, pathway \u003cem\u003ep\u003c/em\u003e\u0026lt;0.01. F) Enriched GO terms from proteins downregulated with AN-R vs AN , pathway \u003cem\u003ep\u003c/em\u003e\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8079717/v1/1ac4c557cf56bb661f75e2f2.jpg"},{"id":96251778,"identity":"da3d4ff5-9ce0-42c6-9ef1-1ceeaf1363d5","added_by":"auto","created_at":"2025-11-19 07:40:01","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":152064,"visible":true,"origin":"","legend":"\u003cp\u003eA-F) Animal-matched correlation between muscle vs serum protein expression.\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8079717/v1/40132f40afbf868f5f17d026.jpg"},{"id":96157763,"identity":"a988a56b-ebfa-467d-9498-35b37432bd53","added_by":"auto","created_at":"2025-11-18 08:37:00","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":167680,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation between muscle MSRB1 protein expression and A) maximal plantar flexor strength. B) rear paw grip, and C) gastrocnemius mass. Correlation between serum TRAIL protein expression and D) maximal plantarflexor strength, E) rear paw grip strength, and F) gastrocnemius mass\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8079717/v1/8b3b19f80f4227a0761d1092.jpg"},{"id":97250334,"identity":"0c328c61-edff-47cb-b277-69abc482b92f","added_by":"auto","created_at":"2025-12-02 13:14:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1499094,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8079717/v1/d37d8b64-b9c5-4dab-90d3-e48390c62661.pdf"},{"id":96251866,"identity":"63229f65-346c-4c5c-a03e-b5e8d122d234","added_by":"auto","created_at":"2025-11-19 07:40:09","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":114054,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Figure 1 Legend. \u003c/strong\u003eCorrelation between muscle NDK7 protein expression and A) maximal plantarflexor strength. B) rear paw grip, C) gastrocnemius CSA, D) gastrocnemius mass; Correlation between serum NAB2 protein expression and E) maximal plantarflexor strength, F) rear paw grip, G) gastrocnemius CSA, H) gastrocnemius mass\u003c/p\u003e","description":"","filename":"SupplementFig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8079717/v1/addb3c2b1f97549bc763c511.jpg"},{"id":96157767,"identity":"1c89bdb0-a1df-4fee-8acb-c160d5bcdec8","added_by":"auto","created_at":"2025-11-18 08:37:00","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":210021,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Figure 2 Legend. \u003c/strong\u003eA) Venn diagram comparing up and downregulated proteins between AN vs CON in muscle and serum. B) Venn diagram comparing up and downregulated proteins between AN-R vs AN in muscle and serum. C) Venn diagrams comparing number and % of differentially abundant upregulated proteins in serum between AN-R vs CON and AN-R vs AN. D) Venn diagrams comparing number and % of differentially abundant upregulated proteins in serum between AN-R vs CON and AN-R vs AN.\u003c/p\u003e","description":"","filename":"SupplementFig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8079717/v1/fbf00b1e1216a28ed5ce8368.jpg"}],"financialInterests":"The authors have declared there is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose","formattedTitle":"Divergent alterations in the skeletal muscle and serum proteome in a rodent model of anorexia nervosa and weight recovery","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAnorexia Nervosa (AN) and its associated complications remain a significant public health challenge for clinicians, patients, and their families. Anorexia Nervosa affects\u0026thinsp;~\u0026thinsp;4% of women and 0.5% of men in the United States\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, and these percentages correspond to millions of individuals and billons of dollars in healthcare expenses\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. AN is also one of the most deadly psychiatric disorders; individuals with AN have a 4.5-fold increased mortality risk compared to those without AN\u003csup\u003e6\u003c/sup\u003e. Despite its prevalence and lethality, treatment options for those with AN are limited and often ineffective, resulting in prolonged disease. This sustained disease resistance necessitates novel research avenues to understand the complex interplay of physiology and psychology in this population.\u003c/p\u003e\u003cp\u003eAlong with fat loss, the implications of which largely through leptin have been studied extensively\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, there is a concurrent loss of skeletal muscle during AN. This skeletal muscle loss has been largely uninvestigated across clinical or basic studies of AN. However, recent evidence demonstrates skeletal muscle loss is a significant predictor of morbidity across multiple pathologies such as cancer, aging, others\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. It is therefore plausible that low muscle mass during AN may be an under investigated contributor to the overall pathophysiology of the condition and contribute to treatment challenges. Our group\u0026rsquo;s recent systematic review found\u0026thinsp;~\u0026thinsp;25% lower skeletal muscle mass/strength in those with AN compared to healthy controls, which is not fully recovered after weight restoration (~\u0026thinsp;9% lower) \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Other recent studies have provided some mechanistic insights for low muscle mass in those with AN. For example, our previous works found altered Leukemia Inhibitory Factor Receptor (LIF-R) in the serum of individuals with AN\u003csup\u003e11\u003c/sup\u003e. LIF-R is a key modulator of cancer-associated muscle wasting\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. LIF-R is also one of the few proteins that differentiated individuals with AN from healthy controls. Other recent works suggest acute alterations to both protein synthetic signaling during AN, which are only partially restored with weight recovery\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Emerging evidence therefore suggests that skeletal muscle physiology may be an important contributor to physiological alterations concurrent with AN. Indeed, the myostatin-activin-follistatin pathway has recently been implicated as a key moderator of loss of muscle functional performance in relative energy deficiency in sport (RED-S) \u003csup\u003e14\u003c/sup\u003e. Yet, to our knowledge, in-depth biological evaluation of the skeletal muscle during extreme caloric deprivation characteristic of AN does not exist.\u003c/p\u003e\u003cp\u003eCurrently, most of our biological understanding of physiological alterations during AN are based solely on blood-based biomarkers. While these markers are clearly important and less invasive than alternative approaches, more direct assessment of skeletal muscle alterations are necessary to fully understand the biological implications of skeletal muscle health during AN and following weight restoration. In this study, we leveraged our recently published rodent model of AN and recovery to evaluate proteomic alterations within the skeletal muscle and the serum during AN and following weight restoration. We find broad proteomic alterations to the skeletal muscle that are not resolved following weight restoration. Moreover, we find proteomic changes in the skeletal muscle are unique in the serum, but that conserved biomarkers of systemic and skeletal muscle disruption during AN may exist. Taken together, these data strongly imply physiological and musculoskeletal consequences of AN are longer lasting and more difficult to identify than previously anticipated.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cem\u003eAnimals Experiments\u003c/em\u003e: All procedures were approved by the Beth Israel Deaconess Medical Center Institutional Animal Care and Use Committee (AUP: 009-2022). We used our recently refined model of AN and AN recovery, which includes group housing rodents for the duration of simulated AN instead of single housing\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. These rodents are subsets of previously reported studies from our laboratory\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. All rats were housed in a temperature and humidity-controlled facility kept at ~\u0026thinsp;23\u003csup\u003eo\u003c/sup\u003eC with a 12:12 hour light:dark cycle. At 8 weeks of age, female Sprague Dawley rats began simulated AN protocol, consisting of ~\u0026thinsp;50\u0026ndash;60% food restriction. Food consumption was assessed 3 days prior to initiation of simulated AN to obtain a baseline food measurement. AN rats were fed\u0026thinsp;~\u0026thinsp;8:00 AM each day, wherein they were moved to individual cages for feeding and allowed up to 2 hours to consume food. Rats were then returned to group housing until feeding the following day. Simulated AN occurred for 30 days, afterwards one cohort of rats (AN, n\u0026thinsp;=\u0026thinsp;8) were anesthetized with 2.5% isoflurane and gastrocnemius muscles collected and snap frozen in liquid nitrogen for further analysis. Rats were euthanized while under anesthesia by cardiac puncture. Another cohort of rats was provided food \u003cem\u003ead libitum\u003c/em\u003e after simulated AN to mirror weight recovery (AN-R, n\u0026thinsp;=\u0026thinsp;8). AN-R rats continued recovery interventions for 30 days. Afterwards, rats were anesthetized with isoflurane (~\u0026thinsp;3%), and gastrocnemius muscles collected for further analysis. Control (CON) rats consumed food \u003cem\u003ead libitum\u003c/em\u003e throughout the protocol and were age-matched to AN-R. All gastrocnemius muscles and serum were collected in the fed state (~\u0026thinsp;90 min after feeding) using a standardized amount of food for all groups (~\u0026thinsp;3 grams of food).\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eNerve stimulated force production\u003c/strong\u003e\u003cp\u003eMaximal force production of the plantar flexor muscles (encompassing the gastrocnemius) was completed\u0026thinsp;~\u0026thinsp;24 hours prior to euthanasia and completed similar to our previous reports\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Briefly, rats were anesthetized with ~\u0026thinsp;2.5% isoflurane. Their tibial nerve was then stimulated with electrode needles to plantar flexor muscle contraction. Small twitches (10 Hz) were used to ensure appropriate needle placement; thereafter rats received one maximal stimulation at 200 Hz for 200 ms. Graphs of maximal tetanus were visually inspected to ensure a full tetanic contraction was achieved.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eGrip Strength\u003c/strong\u003e\u003cp\u003eMaximal rear paw grip strength was completed\u0026thinsp;~\u0026thinsp;24 hours prior to euthanasia as we have described\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Briefly, rats were gently restrained by a trained investigator and the two hindlimb paws placed on a grip bar attached to a force transducer. The rat was then gently pulled from the transducer until the rat released both paws. The corresponding force was then recorded. The procedure was repeated three times and the maximal measurement was used for analysis.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eSomaScan\u003c/strong\u003e\u003cp\u003eSerum and skeletal muscle tissue underwent SOMAscan discovery proteomics at the Genomics, Proteomics, Bioinformatics and Systems Biology Center at Beth Israel Deaconess Medical Center. Briefly, the SOMAscan platform is aptamer-based technology that utilizes single-stranded DNA chemically modified to enhance protein binding with high specificity to measure\u0026thinsp;\u0026gt;\u0026thinsp;1,300 proteins\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Samples were prepared following manufacturer\u0026rsquo;s recommended protocols. Data quality control and normalization procedures were followed as per previous publications\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eStatistical and Pathway Analysis\u003c/em\u003e: Phenotypic data were analyzed with SAS (SAS\u0026reg; Studio Release: 3.81, SAS Institute Inc. Cary, NC) with a one-way ANOVA and a Tukey-adjusted post-hoc p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Differentially expressed proteins were determined using SomaLogic\u0026rsquo;s DataDelve\u0026trade; Statistics analytical platform. Three AN samples were found to have significantly divergent proteomic profiles compared to the remainder of the group. Upon further examination of the data, we noted these animals did not lose as much muscle strength or bodyweight compared to other rats within the group. We therefore set those rats aside for subsequent proteomic analysis. Similarly, three CON serum profiles were set aside as outliers due to substantial differences in their proteomic signatures compared to the rest of the group. We analyzed up and down-regulated proteins among three specific comparisons: AN v. CON, AN v. AN-R and CON v. AN-R. To refine which processes are persistently dysregulated, we performed pathway analyses on proteins persistently dysregulated between AN and AN-R with background correction (background: all proteins detected in dataset). No pathways reached significance at adjusted p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, so we used a non-adjusted p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 as our significance threshold. After differentially altered proteins were assessed, we used ShinyGO 0.82, an online web browser platform, for enrichment analyses to evaluate altered Gene Ontology (GO) pathways\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Correlations were assessed with GraphPad Prism Version 10.2.3. All raw and analyzed data for this project can be found on this project\u0026rsquo;s Open Science Framework Page (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://osf.io/vhy8f/overview?view_only=e721e9b44fa0499195e4cab54c6615d3\u003c/span\u003e\u003cspan address=\"https://osf.io/vhy8f/overview?view_only=e721e9b44fa0499195e4cab54c6615d3\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cem\u003eSimulated AN resulted in lower muscle size and strength, which were not fully resolved following weight gain\u003c/em\u003e\u003c/p\u003e\u003cp\u003eSimulated AN resulted in significant differences in bodyweight compared to CON (~\u0026thinsp;36%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e); however, with refeeding bodyweight differences were no longer present between AN-R and CON (~\u0026thinsp;5%, p\u0026thinsp;=\u0026thinsp;0.066, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). AN resulted in lower rear paw grip strength (~\u0026thinsp;40%) which was not restored in AN-R rats compared to CON (~\u0026thinsp;22% lower, p\u0026thinsp;=\u0026thinsp;0.010, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Maximal plantar flexion was ~\u0026thinsp;25% lower in AN rats compared to CON (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). With weight recovery, plantar flexion in AN-R was not different from CON (~\u0026thinsp;9% lower, p\u0026thinsp;=\u0026thinsp;0.105, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Gastrocnemius mass was ~\u0026thinsp;32% lower in AN compared to CON (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and with weight recovery, AN-R was still\u0026thinsp;~\u0026thinsp;12% lower compared to CON (p\u0026thinsp;=\u0026thinsp;0.003, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCON\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAN\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAN-R\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBodyweight (g)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e234.13\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;4.79\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e148.80\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;1.70\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e221.18\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;4.28\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRear Paw Grip (N)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e8.44\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.22\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5.12\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.43\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7.0167\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.23\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMaximal Plantar Flexion (mN)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e120.54\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;2.30\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e90.35\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;4.79\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e109.87\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;2.97\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGastrocnemius Mass (g)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.170\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.0247\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.7919\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.0123\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.0319\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.0361\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"4\"\u003e\u003cem\u003ePhenotypic data of bodyweight and muscle outcome variables. Data are depicted as Mean\u0026thinsp;+\u0026thinsp;SEM. Different letters represent statistically different at Tukey adjusted p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. CON\u0026thinsp;=\u0026thinsp;control, AN\u0026thinsp;=\u0026thinsp;anorexia nervosa, AN-R\u0026thinsp;=\u0026thinsp;anorexia nervosa\u0026thinsp;+\u0026thinsp;recovery. N\u0026thinsp;=\u0026thinsp;8/group\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eSimulated AN resulted in large changes to the skeletal muscle proteome, which were not restored following weight gain\u003c/em\u003e\u003c/p\u003e\u003cp\u003eWe sought to examine alterations to the muscle proteome as a potential driver of muscle mass and functional losses during food-restricted anorexia nervosa (AN) and subsequent recovery with re-feeding (AN-R) in the gastrocnemius muscle. With AN compared to CON timepoint, there were 112 upregulated and 256 downregulated differentially abundant proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The top 3 differentially abundant upregulated proteins were PFKM, RBM3 and KCTD13 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The top 3 abundant downregulated proteins were DHP5, FGF8 and ARL14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The remaining top 10 proteins in each direction are shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC. There were only 5 proteins that were altered with AN that returned to baseline with recovery (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), indicating that many of the alterations that occurred during AN were not resolved with weight gain. Instead, we see persistent alteration of the proteome after the recovery phase (AN-R), with 245 upregulated and 377 downregulated proteins compared to CON. We compared the overlap in differentially expressed proteins between the AN and AN-R proteomes and found 65 of 112 (58%) proteins upregulated with AN remained upregulated at the AN-R timepoint relative to CON, and 201 of 256 proteins (78%) downregulated with AN remained downregulated at AN-R timepoint (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Some of the top upregulated pathways included protein-DNA complex assembly, apoptotic signaling related to p53, and glycoside metabolic processing (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eDownregulated pathways related to cation transport, muscle satellite cell proliferation, calcium binding, synapse organization and others (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Persistent repression of proteins related to the synapse, and binding/transport of cations which are essential for muscle contraction could perhaps explain why volitional muscle strength was not recovered in AN-R rats compared to CON.\u003c/p\u003e\u003cp\u003e\u003cem\u003eSimulated AN resulted in changes to serum proteome; however, AN-R was more altered compared to AN\u003c/em\u003e\u003c/p\u003e\u003cp\u003eWe next wanted to determine if the serum proteome reflects the muscle proteome during AN and AN-R to identify potential biomarkers of muscle function and size that could be obtained from blood, as muscle biopsies are quite invasive. Only 15 proteins were differentially expressed after AN compared to CON (2 up and 13 down, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). No proteins were up or downregulated in muscle and serum simultaneously at any time point (Supplement A and B). Only one protein, GLTP, a glycolipid transfer protein, was upregulated in muscle while downregulated in serum.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eMost changes in the serum proteome occurred with refeeding, with 317 and 102 upregulated and downregulated proteins, respectively, in AN-R versus AN (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). We also performed pathway analyses on those proteins (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Upregulated GO terms relate to calcium ion binding and TRAIL (TNF-related apoptosis-induced ligand)-activated apoptotic signaling, apoptotic signaling (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Downregulated GO terms related to telomere maintenance, Cajal body localization, and endopeptidase activity and others including skeletal muscle tissue regeneration (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF)\u003c/p\u003e\n\u003ch3\u003eSeveral proteins are correlated between the skeletal muscle and serum across different experimental timepoints\u003c/h3\u003e\n\u003cp\u003eWhile general patterns between the muscle and serum proteome were not present, we next sought to evaluate if any individual proteins showed convergent patterns between muscle and serum samples. We used animal-matched normalized proteomic values for each sample across timepoints to calculate correlations between muscle and serum. Once significant correlations were identified, individual proteins were further evaluated to assess if correlations were being driven by outliers. We found six proteins that had a significant correlation between muscle and serum (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-F). Of those six, NDK7 had the strongest correlation between muscle and serum (r\u0026thinsp;=\u0026thinsp;0.847, p\u0026thinsp;=\u0026thinsp;0.0017, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), followed by NAB2 (r\u0026thinsp;=\u0026thinsp;0.797, p\u0026thinsp;=\u0026thinsp;0.0007, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), and TRAIL (TNFSF10, r\u0026thinsp;=\u0026thinsp;0.642, p\u0026thinsp;=\u0026thinsp;0.0014, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eOf proteins that correlate between biological samples, several also correlated with muscle function\u003c/h3\u003e\n\u003cp\u003eWe conducted correlations on a few key proteins in muscle and serum in relation to functional outcomes. MSRB1 (methionine sulfoxide reductase B1) was one of the few proteins that was altered with AN and restored in AN-R. Moreover, previous work suggests selenium metabolism is important moderator for disease severity during AN\u003csup\u003e24\u003c/sup\u003e and may be a key driver of muscle health\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Therefore, we first correlated muscle MSRB1 to key metrics of muscle health and performance. Overall MSRB1 had strong correlations with maximal plantar flexion (r\u0026thinsp;=\u0026thinsp;0.7965, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), rear paw grip strength (r\u0026thinsp;=\u0026thinsp;0.7273, p\u0026thinsp;=\u0026thinsp;0.002), muscle fiber cross-sectional area (r\u0026thinsp;=\u0026thinsp;0.5902, p\u0026thinsp;=\u0026thinsp;0.0048), and gastrocnemius mass (r\u0026thinsp;=\u0026thinsp;0.7441, p\u0026thinsp;=\u0026thinsp;0.0001, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), overall implying some of the proteomic signatures from our rats corresponded to functional outcomes.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eGiven the previous correlations between serum and muscle proteins for NDK7 and NAB2, we next evaluated if the serum content of these proteins correlated to functional outcomes. Overall, neither protein strongly correlated with any functional parameters (Supplementary Fig.\u0026nbsp;2), suggesting serum content of these proteins does not appear related to muscle function. However, TRAIL serum content did correlate to muscle functional outcomes including maximal plantar flexion (r\u0026thinsp;=\u0026thinsp;0.7308, p\u0026thinsp;=\u0026thinsp;0.0006), rear paw grip strength (r\u0026thinsp;=\u0026thinsp;0.8371, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), muscle fiber cross-sectional area (r\u0026thinsp;=\u0026thinsp;0.6876, p\u0026thinsp;=\u0026thinsp;0.0016), and gastrocnemius mass (r\u0026thinsp;=\u0026thinsp;0.8333, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). Together, TRAIL was the only protein that correlated between muscle and serum and correlated with functional parameters of muscle strength and size.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study we utilized a novel proteomic technique to evaluate the proteomic signature of both skeletal muscle and serum in a rodent model of AN and subsequent weight recovery. We found significant alterations to the skeletal muscle proteome during AN that are not resolved with weight recovery. These broad proteomic perturbations mirror phenotypic alterations within the skeletal muscle, with sustained alterations to muscle function despite weight gain. However, within the serum of these same animals, we find relatively minor proteomic changes in AN rats and instead find dramatic alterations in the proteomic signature of the AN-R rats compared to healthy controls. Moreover, the broad proteomic changes within the serum are not generally reflected in the skeletal muscle proteomic changes. In aggregate, these data demonstrate the profound alterations to physiology during AN and suggest prolonged physiological alterations due to AN.\u003c/p\u003e\u003cp\u003eOne of the initial goals of this study was to evaluate overlap of serum and skeletal muscle proteomic signatures in our model, potentially allowing for a less invasive and faster (compared to maximal muscle testing) method to evaluate skeletal muscle health. Broadly, we find the proteomic signatures of serum and muscle did not overlap. Specifically, within skeletal muscle we note large changes to the proteome during AN, many of which are not resolved with weight recovery. Contrastingly, within the serum, we find relatively modest changes in the proteome during AN compared to CON, yet with weight recovery (AN-R) there were large alterations compared to either AN or CON. There are a few reasons the skeletal muscle proteome may not fully reflect circulating proteome. First, the serum proteome reflects paracrine activity of the skeletal muscle, but also of all other organs within the body. Given that AN is a whole-body pathology, it is likely other tissues (liver, fat, etc.) are also experiencing profound proteomic modifications. The combination of all of these tissue-specific proteomic changes and corresponding alterations to paracrine activity of these tissues would result in systemic proteomic changes that do not directly mirror tissue-specific proteomic changes. Additionally, the skeletal muscle proteomic changes may reflect alterations to tissue specific biology or changes to the paracrine activity of the skeletal muscle, at present we cannot directly evaluate which transformations within the skeletal muscle tissue are directly altering myokine release from the skeletal muscle and this warrants further investigation. Regardless, our findings suggest proteomic alterations to skeletal muscle and blood are unique during AN and following weight restoration, implying more nuanced or novel techniques will likely be necessary to fully understand the biological ramifications of AN and to develop possible future biomarkers of muscle health that do not require an invasive muscle biopsy or maximal muscle strength assessment.\u003c/p\u003e\u003cp\u003eOur data seemingly contrasts other proteomic studies evaluating the serum proteomic alterations during the low weight phase of AN. These differences may in part be due to controlling for multiple comparisons with the SomaScan platform that evaluates significantly more proteins compared to other proteomic platforms as well as the low number of animals per group. However, prior studies in individuals with AN have tended to suggest inflammatory/cytokine signaling and complement may be key pathways in the etiology of AN\u003csup\u003e11, 26\u0026ndash;28\u003c/sup\u003e. Moreover, a recent study evaluating proteomic alterations during acute starvation found similar signaling pathways such as inflammation and complement signaling\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, suggesting this may be an important physiological alteration that is conserved during severe energy restriction regardless of psychological status. Within our data sets, we also do note some signaling changes that may also support this mechanism such as TRAIL cytokine signaling within the serum and muscle (discussed further below) as well as IL-1Beta and T-helper cell differentiation within the skeletal muscle. However, our data strongly suggests, broad proteomic changes within the skeletal muscle are not reflected in the serum either during acute starvation or during weight restoration. These findings provide context for previous studies and future studies on the reliability of correlating serum findings with skeletal muscle health and further highlight the need for new technologies to non-invasively assess muscle health.\u003c/p\u003e\u003cp\u003eWithin the skeletal muscle, many of the top up-regulated and down-regulated proteins during AN are relatively novel in the skeletal muscle literature. These new discoveries may open new avenues to evaluate the relative roles of these proteins in skeletal muscle biology and how they affect systemic health. For example, PFKM is known to be the muscle-specific isoform of PFK, a key glycolytic enzyme. Given limited glucose availability is inherent to AN, the greater abundance of PFKM is a bit paradoxical; however, this specific isoform of PFK may have hitherto unknow roles in muscle biology. Additionally, KCTD13 has previously been reported as a possible genetic contributor to the development of neuropsychiatric diseases\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Genetic deletion of KCTD13 inhibited DNA synthesis and proliferation in human-induced pluripotent stem cells\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. RBM3 is the only top differentially expressed proteins that has previously been reported in skeletal muscle literature. Specifically, the protein is known to be induced in cold environments such as hibernating mammals\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. More recently, mechanistic studies of RBM3 have demonstrated it is sufficient to improve mitochondrial metabolism and myoblast differentiation\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Rodent studies have found RBM3 is elevated during physiological caloric restriction during aging, which is thought to contribute to caloric restriction\u0026rsquo;s longevity effects\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Since there was greater RBM3 content in the present study, it is difficult to fully interpret its role in skeletal muscle physiology and the nuances of RBM3 in relation to pathological (ie starvation/AN) vs. physiological caloric restriction is warranted.\u003c/p\u003e\u003cp\u003eDHPS, FGF8, and ARL14 were the top differentially abundant down-regulated proteins in AN skeletal muscle compared to CON. DHPS is a key protein necessary for activation of eIF5A, which appears necessary for satellite cell activation and presumably muscle growth\u003csup\u003e\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Moreover, loss of DHPS in zebrafish resulted in significant defects in pancreatic growth, accompanied by changes in gene expression related to mRNA translation, neurogenesis, and stress pathways\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Similarly, FGF8 is known to facilitate myogenesis and prevent fat accumulation within skeletal muscle\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. ARL14 has only been reported within the context of cancer\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, so its role in skeletal muscle biology remains unclear. In aggregate, considering the top down-regulated proteins are related to muscle growth and remain depressed during AN-R, these results strongly suggest muscle growth pathways are severely altered during AN and simple weight recovery (with duration matching length of starvation) is not sufficient to restore these pathways.\u003c/p\u003e\u003cp\u003eBroadly, the overall patterns in the proteome of the skeletal muscle compared to serum did not have significant overlap, suggesting that broadly serum protein markers are likely not viable methods to estimate muscle health in humans. However, three proteins broadly have values that correlated within the serum and the skeletal muscle, NDK7, NAB2, and TRAIL. While NDK7 and NAB2 overall had strong correlations within the serum and skeletal muscle (r\u0026thinsp;=\u0026thinsp;0.847 and r\u0026thinsp;=\u0026thinsp;0.798 respectively), they did not generally correlate with either muscle mass or muscle function outcomes (r range\u0026thinsp;=\u0026thinsp;0.328\u0026ndash;0.541). Suggesting neither of those proteins are necessarily indicative of muscle health per se.\u003c/p\u003e\u003cp\u003eTRAIL content in the skeletal muscle and serum correlated (r\u0026thinsp;=\u0026thinsp;0.641) and TRAIL content within the serum correlated strongly with muscle size and functional outcomes (range\u0026thinsp;=\u0026thinsp;0.688\u0026ndash;0.837). Functionally, TRAIL is a cytokine within the TNF family of cytokines\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Within skeletal muscle, the exact role for TRAIL within skeletal muscle health is not fully clear, with some studies finding TRAIL to negatively regulate muscle differentiation\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, whereas others have indicated TRAIL is an important moderator of muscle differentiation and Akt signaling\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Given the known phenotypic alterations to muscle health in this particular model, the latter explanation of TRAIL activity seems likely; however, more mechanistic studies are likely necessary to fully understand the relevance of TRAIL to muscle biology and subsequent health. Regardless, these correlational data suggest serum TRAIL content may be a viable candidate for estimating muscle health during AN and subsequent recovery in human patients. Previous works in patients with AN have also found lower serum TRAIL content\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, overall providing additional clinical robustness to our current findings. Therefore, TRAIL content may be a viable candidate to non-invasively estimate muscle health in patients with AN or recovering from AN and additional studies to directly assess this hypothesis in humans are warranted.\u003c/p\u003e\u003cp\u003eThere are a few limitations of this study that should be acknowledged. We are likely not fully recapitulating the nuances of a complex psychological disorder such as AN with our rodent model. However, as some of our results in rats appear to replicate some results found in humans, our rodent model appears to at least mimic the physiological responses to severe caloric restriction inherent to AN. Additionally, for this study we utilized a different proteomics technology (SomaScan) compared to other previously reported proteomic based analysis (O-link or GC-MS based platforms). These differences between methods can make direct comparisons between studies difficult. For example, due to the broad range of the SomaScan platform (\u0026gt;\u0026thinsp;7000 proteins identified) and the mathematical need to correct for multiple comparisons, our analysis may have omitted some proteins that other analysis with smaller protein identification ranges may have identified as significant. Moreover, we cannot account for possible differences in the serum or skeletal muscle proteome based on age. Our CON and AN-R rats are four weeks older than our AN rats in order to age-match these two groups. Therefore, it is possible that some differences may be due to maturation of the rats during the course of the study. Similarly, our results reflect a duration of recovery matching the duration of simulated AN. We cannot determine if proteomic changes are persistent in perpetuity or if these proteomic changes would be resolved given additional recovery time. Finally, a proteomics analysis of the selected tissues only reflects the protein content within that specific tissue and does not indicate what cell types these proteins are being synthesized from (multiple tissues can contribute to the proteomic signature in the serum and there are multiple different cell types within skeletal muscle). These limitations can be addressed in future studies using more targeted proteomic or transcriptomic approaches, such as single-fiber approaches or single-cell RNA sequencing.\u003c/p\u003e\u003cp\u003eIn this study we evaluated the proteome of skeletal muscle and serum from a rodent model of AN and subsequent weight recovery. We find the skeletal muscle proteome is largely altered during AN and remains altered during weight restoration. Moreover, the serum proteome is largely altered during weight restoration. These results appear to suggest physiological responses to AN and weight recovery are longer-lasting than previously anticipated. Moreover, we find changes to the skeletal muscle and serum proteome do not broadly overlap, with a few exceptions. Specifically, TRAIL content appears to correlate across tissues and functional outcomes, suggesting assessment of this protein may serve as a viable biomarker in the future and/or may be a possible physiological moderator of muscle derangements during AN. Future research should seek to further clarify the relationship between TRAIL (circulating and muscle specific) and muscle functional outcomes in human patients with AN. If the results from our current investigation are consistent in humans, TRAIL content may offer a novel tool to evaluate physiological recovery from AN that is independent of psychological assessments.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of Interest:\u0026nbsp;\u003c/strong\u003eThe authors declare no conflicts of interests related to this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u003c/strong\u003e Raw data and associated statistical code are available at our Open Science Framework page for this project at: https://osf.io/vhy8f/overview?view_only=e721e9b44fa0499195e4cab54c6615d3\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e: This study was supported by the Beth Israel Deaconess Medical Center Translational Spark Hubs Grant, (M.E.R/C.M./L.B.), NIH grant K12HD051959 (M.E.R.) and the Arkansas Integrative Metabolic Research Center Pilot Grant (M.E.R.) as part of the AIMRC Center for Biomedical Research Excellence (COBRE) grant (5P20GM139768). We would like to thank Eliza Garland, Sheridyn Pinkham, Madisyn Hancock, and Sophie Jalkut for their assistance with this project.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSmink FR, van Hoeken D, Hoek HW. Epidemiology, course, and outcome of eating disorders. \u003cem\u003eCurr Opin Psychiatry\u003c/em\u003e 2013; \u003cstrong\u003e26\u003c/strong\u003e(6)\u003cstrong\u003e: \u003c/strong\u003e543-548.\u003c/li\u003e\n\u003cli\u003eAE vE, D vH, HW H. Incidence, prevalence and mortality of anorexia nervosa and bulimia nervosa. \u003cem\u003eCurrent opinion in psychiatry\u003c/em\u003e 2021; \u003cstrong\u003e34\u003c/strong\u003e(6).\u003c/li\u003e\n\u003cli\u003eMicali N, Martini MG, Thomas JJ, Eddy KT, Kothari R, Russell E\u003cem\u003e et al.\u003c/em\u003e Lifetime and 12-month prevalence of eating disorders amongst women in mid-life: a population-based study of diagnoses and risk factors. \u003cem\u003eBMC Med\u003c/em\u003e 2017; \u003cstrong\u003e15\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eMitchell JE, Myers T, Crosby R, O\u0026rsquo;Neill G, Carlisle J, Gerlach S. Health Care Costs in Patients with Eating Disorders. \u003cem\u003eInt J Eat Disord\u003c/em\u003e 2009; \u003cstrong\u003e42\u003c/strong\u003e(6)\u003cstrong\u003e: \u003c/strong\u003e571-574.\u003c/li\u003e\n\u003cli\u003ede Oliveira C, Colton P, Cheng J, Olmsted M, Kurdyak P. The direct health care costs of eating disorders among hospitalized patients: A population-based study. \u003cem\u003eInt J Eat Disord\u003c/em\u003e 2017; \u003cstrong\u003e50\u003c/strong\u003e(12)\u003cstrong\u003e: \u003c/strong\u003e1385-1393.\u003c/li\u003e\n\u003cli\u003eS\u0026oslash;eby M, Gribsholt SB, Clausen L, Richelsen B. Overall and cause-specific mortality in anorexia nervosa; impact of psychiatric comorbidity and sex in a 40-year follow-up study. \u003cem\u003eThe International journal of eating disorders\u003c/em\u003e 2024; \u003cstrong\u003e57\u003c/strong\u003e(9).\u003c/li\u003e\n\u003cli\u003eKhan SM, Hamnvik OP, Brinkoetter M, Mantzoros CS. Leptin as a modulator of neuroendocrine function in humans. \u003cem\u003eYonsei medical journal\u003c/em\u003e 2012; \u003cstrong\u003e53\u003c/strong\u003e(4).\u003c/li\u003e\n\u003cli\u003eChrysafi P, Perakakis N, Farr OM, et al\u003cem\u003e.\u003c/em\u003e Leptin alters energy intake and fat mass but not energy expenditure in lean subjects. \u003cem\u003eNature communications\u003c/em\u003e 2020; \u003cstrong\u003e11\u003c/strong\u003e(1).\u003c/li\u003e\n\u003cli\u003eZhou HH, Liao Y, Peng Z, Liu F, Wang Q, Yang W. Association of muscle wasting with mortality risk among adults: A systematic review and meta-analysis of prospective studies. \u003cem\u003eJournal of cachexia, sarcopenia and muscle\u003c/em\u003e 2023; \u003cstrong\u003e14\u003c/strong\u003e(4).\u003c/li\u003e\n\u003cli\u003eRosa-Caldwell ME, Eddy KT, Rutkove SB, Breithaupt L. Anorexia nervosa and muscle health: A systematic review of our current understanding and future recommendations for study. \u003cem\u003eThe International journal of eating disorders\u003c/em\u003e 2022.\u003c/li\u003e\n\u003cli\u003eBreithaupt L, Holsen LM, Ji C, et al\u003cem\u003e.\u003c/em\u003e Identification of State Markers in Anorexia Nervosa: Replication and Extension of Inflammation-Associated Biomarkers Using Multiplex Profiling. \u003cem\u003eBiological psychiatry global open science\u003c/em\u003e 2024; \u003cstrong\u003e4\u003c/strong\u003e(5).\u003c/li\u003e\n\u003cli\u003eKandarian SC, Nosacka RL, Delitto AE, \u003cem\u003eet al.\u003c/em\u003e Tumour-derived leukaemia inhibitory factor is a major driver of cancer cachexia and morbidity in C26 tumour-bearing mice. \u003cem\u003eJournal of cachexia, sarcopenia and muscle\u003c/em\u003e 2018; \u003cstrong\u003e9\u003c/strong\u003e(6).\u003c/li\u003e\n\u003cli\u003eRosa-Caldwell M, Breithaupt L, Kaiser U, Muhyudin R, Rutkove S. Changes in muscle strength and moderators of protein turnover in a rodent model of anorexia nervosa and recovery. \u003cem\u003eJournal of Nutritional Physiology\u003c/em\u003e 2025; \u003cstrong\u003e4\u003c/strong\u003e: 100010.\u003c/li\u003e\n\u003cli\u003eAngelidi AM, Stefanakis K, Chou SH, \u003cem\u003eet al.\u003c/em\u003e Relative Energy Deficiency in Sport (REDs): Endocrine Manifestations, Pathophysiology and Treatments. \u003cem\u003eEndocrine reviews\u003c/em\u003e 2024; \u003cstrong\u003e45\u003c/strong\u003e(5).\u003c/li\u003e\n\u003cli\u003eRosa-Caldwell, E. M, Breithaupt, L., Kaiser, B. U\u003cem\u003e et al.\u003c/em\u003e A refined rodent model of anorexia nervosa: Simulating state-specific effects of caloric restriction and weight restoration. \u003cem\u003ePhysiological Reports\u003c/em\u003e 2024; \u003cstrong\u003e12: \u003c/strong\u003ee16092.\u003c/li\u003e\n\u003cli\u003eRosa-Caldwell ME, Mortreux M, Wadhwa A, et al. Sex differences in muscle health in simulated micro- and partial-gravity environments in rats. Sports Med Health Sci. 2023;\u003cstrong\u003e5\u003c/strong\u003e(4):319-328.\u003c/li\u003e\n\u003cli\u003eRosa-Caldwell ME, Mortreux M, Wadhwa A, et al. Influence of gonadectomy on muscle health in micro- and partial-gravity environments in rats. J Appl Physiol (1985). 2023;\u003cstrong\u003e134\u003c/strong\u003e(6):1438-1449.\u003c/li\u003e\n\u003cli\u003eGold L, Ayers D, Bertino J \u003cem\u003eet al.\u003c/em\u003e Aptamer-based multiplexed proteomic technology for biomarker discovery. \u003cem\u003ePloS one\u003c/em\u003e 2010; \u003cstrong\u003e5\u003c/strong\u003e(12).\u003c/li\u003e\n\u003cli\u003eRohloff JC, Gelinas AD, Jarvis TC,\u003cem\u003e et al.\u003c/em\u003e Nucleic Acid Ligands With Protein-like Side Chains: Modified Aptamers and Their Use as Diagnostic and Therapeutic Agents. \u003cem\u003eMolecular therapy Nucleic acids\u003c/em\u003e 2014; \u003cstrong\u003e3\u003c/strong\u003e(10).\u003c/li\u003e\n\u003cli\u003eCandia J, Cheung F, Kotliarov Y, \u003cem\u003eet al.\u003c/em\u003e Assessment of Variability in the SOMAscan Assay. \u003cem\u003eScientific reports\u003c/em\u003e 2017; \u003cstrong\u003e7\u003c/strong\u003e(1).\u003c/li\u003e\n\u003cli\u003eCandia J, Daya GN, Tanaka T, Ferrucci L, Walker KA. Assessment of variability in the plasma 7k SomaScan proteomics assay. \u003cem\u003eScientific reports\u003c/em\u003e 2022; \u003cstrong\u003e12\u003c/strong\u003e(1).\u003c/li\u003e\n\u003cli\u003eGe SX, Jung D, Yao R. ShinyGO: a graphical gene-set enrichment tool for animals and plants. \u003cem\u003eBioinformatics (Oxford, England)\u003c/em\u003e 2020; \u003cstrong\u003e36\u003c/strong\u003e(8).\u003c/li\u003e\n\u003cli\u003eGene Ontology Consortium, Aleksander SA, Balhoff J, \u003cem\u003eet al.\u003c/em\u003e The Gene Ontology knowledgebase in 2023. \u003cem\u003eGenetics\u003c/em\u003e 2023; \u003cstrong\u003e224\u003c/strong\u003e(1).\u003c/li\u003e\n\u003cli\u003eStrumila R, Lengvenyte A, Olie E, \u003cem\u003eet al.\u003c/em\u003e Selenium deficiency is associated with disease severity, disrupted reward processing, and increased suicide risk in patients with Anorexia Nervosa. \u003cem\u003ePsychoneuroendocrinology\u003c/em\u003e 2022; \u003cstrong\u003e140\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eWang Q, Huang J, Zhu K, Zuo W. Selenium and Skeletal Muscle Health in Sports Nutrition. \u003cem\u003eNutrients\u003c/em\u003e 2025; \u003cstrong\u003e17\u003c/strong\u003e(11).\u003c/li\u003e\n\u003cli\u003eSamodova D, Hoel A, Hansen TH,\u003cem\u003e et al.\u003c/em\u003e Plasma proteome profiling reveals metabolic and immunologic differences between Anorexia Nervosa subtypes. \u003cem\u003eMetabolism: clinical and experimental\u003c/em\u003e 2024; \u003cstrong\u003e152\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eAdams DM, Reay WR, Cairns MJ. Multiomic prioritisation of risk genes for anorexia nervosa. \u003cem\u003ePsychological medicine\u003c/em\u003e 2023; \u003cstrong\u003e53\u003c/strong\u003e(14).\u003c/li\u003e\n\u003cli\u003eNilsson IAK, Millischer V, G\u0026ouml;teson A, \u003cem\u003eet al.\u003c/em\u003e Aberrant inflammatory profile in acute but not recovered anorexia nervosa. \u003cem\u003eBrain, behavior, and immunity\u003c/em\u003e 2020; \u003cstrong\u003e88\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eCommissati S, Cagigas ML, Masedunskas A, \u003cem\u003eet al.\u003c/em\u003e Prolonged fasting promotes systemic inflammation and platelet activation in humans: A medically supervised, water-only fasting and refeeding study. \u003cem\u003eMolecular metabolism\u003c/em\u003e 2025; \u003cstrong\u003e96\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eKizner V, Naujock M, Fischer S, \u003cem\u003eet al.\u003c/em\u003e CRISPR/Cas9-mediated Knockout of the Neuropsychiatric Risk Gene KCTD13 Causes Developmental Deficits in Human Cortical Neurons Derived from Induced Pluripotent Stem Cells. \u003cem\u003eMolecular neurobiology\u003c/em\u003e 2020; \u003cstrong\u003e57\u003c/strong\u003e(2).\u003c/li\u003e\n\u003cli\u003eZhu X, B\u0026uuml;hrer C, Wellmann S. Cold-inducible proteins CIRP and RBM3, a unique couple with activities far beyond the cold. \u003cem\u003eCellular and molecular life sciences : CMLS\u003c/em\u003e 2016; \u003cstrong\u003e73\u003c/strong\u003e(20).\u003c/li\u003e\n\u003cli\u003eDey P, Rajalaxmi S, Saha P, \u003cem\u003eet al.\u003c/em\u003e Cold-shock proteome of myoblasts reveals role of RBM3 in promotion of mitochondrial metabolism and myoblast differentiation. \u003cem\u003eCommunications biology\u003c/em\u003e 2024; \u003cstrong\u003e7\u003c/strong\u003e(1).\u003c/li\u003e\n\u003cli\u003eHettinger ZR, Confides AL, Vanderklish PW, Sidhom S, Masternak MM, Dupont-Versteegden EE. Skeletal muscle RBM3 expression is associated with extended lifespan in Ames Dwarf and calorie restricted mice. \u003cem\u003eExperimental gerontology\u003c/em\u003e 2021; \u003cstrong\u003e146\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eConjard-Duplany A, Osseni A, Lamboux A,\u003cem\u003e et al.\u003c/em\u003e Muscle mTOR controls iron homeostasis and ferritinophagy via NRF2, HIFs and AKT/PKB signaling pathways. \u003cem\u003eCellular and molecular life sciences : CMLS\u003c/em\u003e 2025; \u003cstrong\u003e82\u003c/strong\u003e(1).\u003c/li\u003e\n\u003cli\u003eZhang Q, Han W, Wu R, \u003cem\u003eet al.\u003c/em\u003e Spermidine-eIF5A axis is essential for muscle stem cell activation via translational control. \u003cem\u003eCell discovery\u003c/em\u003e 2024; \u003cstrong\u003e10\u003c/strong\u003e(1).\u003c/li\u003e\n\u003cli\u003eKar RK, Hanner AS, Starost MF, \u003cem\u003eet al.\u003c/em\u003e Neuron-specific ablation of eIF5A or deoxyhypusine synthase leads to impairments in growth, viability, neurodevelopment, and cognitive functions in mice. \u003cem\u003eThe Journal of biological chemistry\u003c/em\u003e 2021; \u003cstrong\u003e297\u003c/strong\u003e(5).\u003c/li\u003e\n\u003cli\u003eAnderson CM, Kulkarni A, Maier B,\u003cem\u003e et al.\u003c/em\u003e Hypusinated and unhypusinated isoforms of the translation factor eIF5A exert distinct effects in models of pancreas development and function. \u003cem\u003eThe Journal of biological chemistry\u003c/em\u003e 2025; \u003cstrong\u003e301\u003c/strong\u003e(2).\u003c/li\u003e\n\u003cli\u003eOtsuka T, Kan HM, Mengsteab PY, Tyson B, Laurencin CT. Fibroblast growth factor 8b (FGF-8b) enhances myogenesis and inhibits adipogenesis in rotator cuff muscle cell populations in vitro. \u003cem\u003eProceedings of the National Academy of Sciences of the United States of America\u003c/em\u003e 2024; \u003cstrong\u003e121\u003c/strong\u003e(1).\u003c/li\u003e\n\u003cli\u003eZhang B, Xu A, Wu D, \u003cem\u003eet al.\u003c/em\u003e ARL14 as a Prognostic Biomarker in Non-Small Cell Lung Cancer. \u003cem\u003eJournal of inflammation research\u003c/em\u003e 2021; \u003cstrong\u003e14\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eWiley SR, Schooley K, Smolak PJ\u003cem\u003e et al.\u003c/em\u003e Identification and characterization of a new member of the TNF family that induces apoptosis. \u003cem\u003eImmunity\u003c/em\u003e 1995; \u003cstrong\u003e3\u003c/strong\u003e(6).\u003c/li\u003e\n\u003cli\u003eKim D, Singh N, Waldemer-Streyer RJ, Yoon MS, Chen J. Muscle-derived TRAIL negatively regulates myogenic differentiation. \u003cem\u003eExperimental cell research\u003c/em\u003e 2020; \u003cstrong\u003e394\u003c/strong\u003e(1).\u003c/li\u003e\n\u003cli\u003eToffoli B, Tonon F, Tisato V,\u003cem\u003e et al.\u003c/em\u003e TRAIL/DR5 pathway promotes AKT phosphorylation, skeletal muscle differentiation, and glucose uptake. \u003cem\u003eCell death \u0026amp; disease\u003c/em\u003e 2021; \u003cstrong\u003e12\u003c/strong\u003e(12).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8079717/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8079717/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAnorexia nervosa (AN) is a severe psychiatric disorder characterized by prolonged caloric restriction and significant weight loss, including loss of skeletal muscle. However, physiological changes during AN and following weight restoration are poorly understood, especially skeletal muscle physiology and how it relates to systemic physiology. In this study, we utilized a rodent model of AN and weight gain to investigate proteomic changes across the skeletal muscle and serum and how these proteomic alterations relate to muscle functional outcomes. Using SOMAscan proteomics, we identified extensive alterations in the skeletal muscle proteome during 30 days of simulated AN, with the majority of these changes persisting after weight restoration. Muscle mass and strength were not fully recovered after weight restoration, aligning with sustained dysregulation of proteins related to apoptosis, calcium handling, and satellite cell proliferation in skeletal muscle. By contrast, serum proteomic changes were modest during AN but markedly altered and largely distinct from muscle during recovery. TRAIL (TNF-related apoptosis-inducing ligand) emerged as a potential non-invasive biomarker of muscle health as TRAIL content correlated between muscle and serum as well as muscle functional assessments. Proteomic biomarkers of muscle versus systemic AN are largely unique, underlining the complexity of this illness and the need for novel analytical approaches; however, TRAIL dysregulation may be a conserved feature across tissues and should be further investigated as potential non-invasive biomarker of muscle health and a possible mechanistic target for AN-induced muscle loss.\u003c/p\u003e","manuscriptTitle":"Divergent alterations in the skeletal muscle and serum proteome in a rodent model of anorexia nervosa and weight recovery","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-18 08:36:55","doi":"10.21203/rs.3.rs-8079717/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ee6a9ec4-ed9b-419b-a940-b9e5a7099775","owner":[],"postedDate":"November 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":57883400,"name":"Health sciences/Diseases/Psychiatric disorders"},{"id":57883401,"name":"Health sciences/Biomarkers/Predictive markers"},{"id":57883402,"name":"Biological sciences/Physiology"}],"tags":[],"updatedAt":"2025-12-02T10:32:31+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-18 08:36:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8079717","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8079717","identity":"rs-8079717","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}