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

Reviewer #1 (Public Review):

Summary:

In this paper, proteomics analysis of the plasma of human subjects that underwent an exercise training regime consisting of a combination of endurance and resistance exercise led to the identification of several proteins that were responsive to exercise training. Confirming previous studies, many exercise-responsive secreted proteins were found to be involved in the extra-cellular matrix. The protein CD300LG was singled out as a potential novel exercise biomarker and the subject of numerous follow-up analyses. The levels of CD300LG were correlated with insulin sensitivity. The analysis of various open-source datasets led to the tentative suggestion that CD300LG might be connected with angiogenesis, liver fat, and insulin sensitivity. CD300LG was found to be most highly expressed in subcutaneous adipose tissue and specifically in venular endothelial cells. In a subset of subjects from the UK Biobank, serum CD300LG levels were positively associated with several measures of physical activity - particularly vigorous activity. In addition, serum CD300LG levels were negatively associated with glucose levels and type 2 diabetes. Genetic studies hinted at these associations possibly being causal. Mice carrying alterations in the CD300LG gene displayed impaired glucose tolerance, but no change in fasting glucose and insulin. Whether the production of CD300LG is changed in the mutant mice is unclear.

Strengths:

The specific proteomics approach conducted to identify novel proteins impacted by exercise training is new. The authors are resourceful in the exploitation of existing datasets to gain additional information on CD300LG.

Weaknesses:

While the analyses of multiple open-source datasets are necessary and useful, they lead to relatively unspecific correlative data that collectively insufficiently advance our knowledge of CD300LG and merely represent the starting point for more detailed investigations. Additional more targeted experiments of CD300LG are necessary to gain a better understanding of the role of CD300LG and the mechanism by which exercise training may influence CD300LG levels. One should also be careful to rely on external data for such delicate experiments as mouse phenotyping. Can the authors vouch for the quality of the data collected?

Reviewer #2 (Public Review):

Summary:

This manuscript from Lee-Odegard et al reports proteomic profiling of exercise plasma in humans, leading to the discovery of CD300LG as a secreted exercise-inducible plasma protein. Correlational studies show associations of CD300LG with glycemic traits. Lastly, the authors query available public data from CD300LG-KO mice to establish a causal role for CD300LG as a potential link between exercise and glucose metabolism. However, the strengths of this manuscript were balanced by the moderate to major weaknesses. Therefore in my opinion, while this is an interesting study, the conclusions remain preliminary and are not fully supported by the experiments shown so far.

Strengths:

(1) Data from a well-phenotyped human cohort showing exercise-inducible increases in CD300LG.

(2) Associations between CD300LG and glucose and other cardiometabolic traits in humans, that have not previously been reported.

(3) Correlation to CD300LG mRNA levels in adipose provides additional evidence for exercise-inducible increases in CD300LG.

Weaknesses:

(1) CD300LG is by sequence a single-pass transmembrane protein that is exclusively localized to the plasma membrane. How CD300LG can be secreted remains a mystery. More evidence should be provided to understand the molecular nature of circulating CD300LG. Is it full-length? Is there a cleaved fragment? Where is the epitope where the o-link is binding to CD300LG? Does transfection of CD300LG to cells in vitro result in secreted CD300LG?

(2) There is a growing recognition of specificity issues with both the O-link and somalogic platforms. Therefore it is critical that the authors use antibodies, targeted mass spectrometry, or some other methods to validate that CD300LG really is increased instead of just relying on the O-link data.

(3) It is insufficient simply to query the IMPC phenotyping data for CD300LG; the authors should obtain the animals and reproduce or determine the glucose phenotypes in their own hands. In addition, this would allow the investigators to answer key questions like the phenotype of these animals after a GTT, whether glucose production or glucose uptake is affected, whether insulin secretion in response to glucose is normal, effects of high-fat diet, and other standard mouse metabolic phenotyping assays.

(4) I was unable to find the time point at which plasma was collected at the 12-week time point. Was it immediately after the last bout of exercise (an acute response) or after some time after the training protocol (trained state)?

Reviewer #3 (Public Review):

Summary:

This manuscript by Liu et al. presents a case that CAPSL mutations are a cause of familial exudative vitreoretinopathy (FEVR). Attention was initially focused on the CAPSL gene from whole exome sequence analysis of two small families. The follow-up analyses included studies in which CAPSL was manipulated in endothelial cells of mice and multiple iterations of molecular and cellular analyses. Together, the data show that CAPSL influences endothelial cell proliferation and migration. Molecularly, transcriptomic and proteomic analyses suggest that CAPSL influences many genes/proteins that are also downstream targets of MYC and may be important to the mechanisms.

Strengths:

This multi-pronged approach found a previously unknown function for CAPSLs in endothelial cells and pointed at MYC pathways as high-quality candidates in the mechanism.

Weaknesses:

Two issues shape the overall impact for me. First, the unreported population frequency of the variants in the manuscript makes it unclear if CAPSL should be considered an interesting candidate possibly contributing to FEVR, or possibly a cause. Second, it is unclear if the identified variants act dominantly, as indicated in the pedigrees. The studies in mice utilized homozygotes for an endothelial cell-specific knockout, leaving uncertainty about what phenotypes might be observed if mice heterozygous for a ubiquitous knockout had instead been studied.

In my opinion, the following scientific issues are specific weaknesses that should be addressed:

(1) Please state in the manuscript the number of FEVR families that were studied by WES. Please also describe if the families had been selected for the absence of known mutations, and/or what percentage lack known pathogenic variants.

(2) A better clinical description of family 3104 would enhance the manuscript, especially the father. It is unclear what "manifested with FEVR symptoms, according to the medical records" means. Was the father diagnosed with FEVR? If the father has some iteration of a mild case, please describe it in more detail. If the lack of clinical images in the figure is indicative of a lack of medical documentation, please note this in the manuscript.

(3) The TGA stop codon can in some instances also influence splicing (PMID: 38012313). Please add a bioinformatic assessment of splicing prediction to the assays and report its output in the manuscript.

(4) More details regarding utilizing a "loxp-flanked allele of CAPSL" are needed. Is this an existing allele, if so, what is the allele and citation? If new (as suggested by S1), the newly generated CAPSL mutant mouse strain needs to be entered into the MGI database and assigned an official allele name - which should then be utilized in the manuscript and who generated the strain (presumably a core or company?) must be described.

(5) The statement in the methods "All mice used in the study were on a C57BL/6J genetic background," should be better defined. Was the new allele generated on a pure C57BL/6J genetic background, or bred to be some level of congenic? If congenic, to what generation? If unknown, please either test and report the homogeneity of the background, or consult with nomenclature experts (such as available through MGI) to adopt the appropriate F?+NX type designation. This also pertains to the Pdgfb-iCreER mice, which reference 43 describes as having been generated in an F2 population of C57BL/6 X CBA and did not designate the sub-strain of C57BL/6 mice. It is important because one of the explanations for missing heritability in FEVR may be a high level of dependence on genetic background. From the information in the current description, it is also not inherently obvious that the mice studied did not harbor confounding mutations such as rd1 or rd8.

(6) In my opinion, more experimental detail is needed regarding Figures 2 and 3. How many fields, of how many retinas and mice were analyzed in Figure 2? How many mice were assessed in Figure 3?

(7) I suggest adding into the methods whether P-values were corrected for multiple tests.