Serum proteomic profiling of physical activity reveals CD300LG as a novel exerkine with a potential causal link to glucose homeostasis

Background: Physical activity has been associated with preventing the development of type 2 diabetes and atherosclerotic cardiovascular disease. However, our understanding of the precise molecular mechanisms underlying these effects remains incomplete and good biomarkers to objectively assess physical activity are lacking. Methods: We analyzed 3072 serum proteins in 26 men, normal weight or overweight, undergoing 12 weeks of a combined strength and endurance exercise intervention. We estimated insulin sensitivity with hyperinsulinemic euglycemic clamp, maximum oxygen uptake, muscle strength, and used MRI/MRS to evaluate body composition and organ fat depots. Muscle and subcutaneous adipose tissue biopsies were used for mRNA sequencing. Additional association analyses were performed in samples from up to 47,747 individuals in the UK Biobank, as well as using 2-sample Mendelian randomization and mice models. Results: Following 12 weeks of exercise intervention, we observed significant changes in 283 serum proteins. Notably, 66 of these proteins were elevated in overweight men and positively associated with liver fat before the exercise regimen, but were normalized after exercise. Furthermore, for 19.7% and 12.1% of the exercise-responsive proteins, corresponding changes in mRNA expression levels in muscle and fat, respectively, were shown. The protein CD300LG displayed consistent alterations in blood, muscle, and fat. Serum CD300LG exhibited positive associations with insulin sensitivity, and to angiogenesis-related gene expression in both muscle and fat. Furthermore, serum CD300LG was positively associated with physical activity and negatively associated with glucose levels in the UK Biobank. In this sample, the association between serum CD300LG and physical activity was significantly stronger in men than in women. Mendelian randomization analysis suggested potential causal relationships between levels of serum CD300LG and fasting glucose, 2-hour glucose after an oral glucose tolerance test, and HbA1c. Additionally, Cd300lg responded to exercise in a mouse model, and we observed signs of impaired glucose tolerance in male, but not female, Cd300lg knockout mice. Conclusion: Our study identified several novel proteins in serum whose levels change in response to prolonged exercise and were significantly associated with body composition, liver fat, and glucose homeostasis. Serum CD300LG increased with physical activity and is a potential causal link to improved glucose levels. CD300LG may be a promising exercise biomarker and a therapeutic target in type 2 diabetes. ### Competing Interest Statement The authors have declared no competing interest. ### Clinical Trial NCT01803568 ### Funding Statement South-Eastern Norway Regional Health Authority, Simon Fougners fund, Diabetesforbundet, Johan Selmer Kvanes legat til forskning og bekjempelse av sukkersyke. This research has been conducted using the UK Biobank resource (Reference 53641). DME is funded by an Australian National Health and Medical Research Council Investigator Grant (APP2017942). GHM is the recipient of an Australian Research Council Discovery Early Career Award (Project number: DE220101226) funded by the Australian Government and supported by the Research Council of Norway (Project grant: 325640 & Mobility grant: 287198). SL is supported by the Novo Nordisk Fonden Excellence Emerging Grant, Endocrinology and Metabolism 2023 (NNF23OC0082123). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The MyoGlu study was conducted as a controlled clinical trial (clinicaltrials.gov: [NCT01803568][1]) and was carried out in adherence to the principles of the Declaration of Helsinki. The study received ethical approval from the National Regional Committee for Medical and Health Research Ethics North in Tromso, Norway, with the reference number 2011/882. All participants provided written informed consent before undergoing any procedures related to the study. The UK biobank has ethical approval from the North West Multi-Centre Research Ethics Committee (MREC), which covers the UK, and all participants provided written informed consent. This particular project from the UK biobank received ethical approval from the Institutional Human Research Ethics committee, University of Queensland (approval number 2019002705). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes mRNA sequencing data from MyoGlu can be found at https://exchmdpmg.medsch.ucla.edu/app/ as well as in GEO:GSE227419. Secretory proteins are available in the MetazSecKB data base at http://proteomics.ysu.edu/secretomes/animal/. The human serum proteomic NAFLD signature is available in the study of Govaere et al 38. Expression profiles in human liver cells are available in the Human Liver Cell Atlas 39. Data obtained from the UK biobank (Olink explore 1536 and measures of physical activity 78 can be found at https://biobank.ndph.ox.ac.uk/ukb/. Glucometabolic outcomes used in MR analyses are available at: https://www.magicinvestigators.org/ 45. Data from the GD-CAT database is available from: https://pipeline.biochem.uci.edu/gtex/demo2/. Mice exercise data are available at https://motrpac-data.org/ and knock-out data at https://www.mousephenotype.org/. CD300LG expression values from a human tissue panel were obtained from Uhlen et al. 42. The single nuclei mRNA sequencing data from human adipose tissue was plotted in Seurat v. 4 by downloading processed data from the Single Cell Portal 43. The data can also be explored at: https://singlecell.broadinstitute.org/single_cell/study/SCP1376/a-single-cell-atlas-of-human-and-mouse-white-adipose-tissue). UK Biobank (https://www.ukbiobank.ac.uk/) data are available to researchers upon application to the individual cohorts via their websites. All other data used are publicly available and referenced according in the main text. For additional details and data inquiries, please contact SL. [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01803568&atom=%2Fmedrxiv%2Fearly%2F2024%2F02%2F12%2F2024.02.10.24302626.atom