Exercise training at different intensities induces heat stress, disrupts barrier function and alters microbiota in the gut of mice

Reviewer #1 (Public review):

Summary:

This article investigated the relationship between different intensities of exercise training and intestinal barrier dysfunction, and further explores the possible mechanisms, including the contribution of stress response, inflammatory response, gut microbiota alterations, and derived metabolites.

Strengths:

This article mainly focused on different aspects of the phenotypes and the morphology of intestinal barrier dysfunction induced by exercise training.

Weaknesses:

This article lacks the verification of the association of causality among various phenotypes and lacks a comprehensive understanding of the underlying mechanisms of how exercise contributes to intestinal barrier dysfunction.

(1) For example, the author claimed that heat shock and ischemia are the causes of intestinal epithelial damage caused by exercise, and it is not only evidenced by detecting the expression of a few regulators, such as HSF and HSP70 after exercise; and by Immunohistochemical analysis of intestinal morphology and inflammation.

(2) Many kinds of intestinal bacteria could produce short-chain fatty acids, such as Faecalibacterium Prausnitzii, did the authors check their abundance in the intestine after exercise training?

(3) How to define exercise intensity? Was VO2 Max testing used in this study?

(4) As the strict control, it is recommended to set 4 groups of exercise training groups: daily vigorous exercise training, daily moderate exercise training, daily vigorous exercise training with intermittent rest days, and daily moderate exercise training with intermittent rest days.

(5) Are there any differences in diet and metabolism between different groups of mice, which may affect the phenotypes, especially the composition and the the diverstiy of gut microbiota?

Reviewer #2 (Public review):

Lian et al. provide novel and exciting findings related to exercise-induced intestinal injury that have many implications for those engaging in any kind of training protocol. The authors continue to provide data demonstrating that different forms of exercise training impart a unique signature to the gut microbiota. The paper is well-written, easy to follow, and contains ample information in all sections. The figures are displayed in a clear and comprehensible format, with elegant images. I do have a few concerns regarding some aspects of the paper listed below, but otherwise, I feel that the authors clearly state their objectives, implement valid methods, and summarize their findings with the appropriate conclusions given their experimental constraints.

(1) The authors performed extensive experiments demonstrating the immediate effects of a bout of exercise on intestinal integrity throughout a 6-week training program. Additionally, the authors go as far as to show that successive exercise sessions appear to augment the observed damage. This is very important and noteworthy data. But I wonder, had the endpoint collections been taken 24 hours+ after the last exercise bout, would the findings be different? My concern is that the 1-hour time point is biased towards seeing more damage. I understand the acute effects of exercise occur and are important to report, but they can be transient, and adaptations ensue. My main concern is that the data shows the onset of the initial damage, but nothing addresses an adaptive or recovery response that could counter the observed exercise-induced intestinal injury. Even metrics such as stool consistency/ pellets per hour/ abnormal defecation measurements could indicate the function of the GI system after exercise and may offer more information related to damage vs recovery.

(2) An additional concern arises with the model of forced treadmill running. It was previously shown that forced treadmill running resulted in more gut damage compared to voluntary wheel running, with or without dextran sodium sulfate-induced colitis (PMID: 23707215). This type of training appears to be very important in initiating damage to the GI. Understanding how much of this is related to the chosen exercise protocol, forced treadmill running, will be very important for future experiments. Exercise intensity has been suggested to be a major factor in exercise-induced intestinal damage. Therefore, the group designated as MOD-EX in this paper may be over the intensity threshold that limits GI damage. The protocols used in this manuscript may be inherently biased towards enhancing exercise-induced GI damage, which is not necessarily negative, especially when a damaging protocol is needed. However, how much this relates to and can be translated to humans is not clear and needs further experimentation.

(3) I think the comparison between groups at the specified time point is important, but I believe additional comparisons should be included that show within-group differences across each time point. For example, in the Mod group, does FITC- dextran change between 4 and 6 weeks? Are there morphological change differences between 2, 4, and 6 weeks within each group? Essentially addressing a progression in damage as a function of the duration of exercise training. The authors clearly show exercise-induced damage to the GI, but we do not know how this damage is handled or if the continuation of exercise continues to reinforce the disruption in the epithelial cells.

(4) The authors describe the purpose of this study as being to identify key regulators of the destruction and reconstruction process of the GI after exercise (introduction lines 128-129). While the authors did sufficient work to describe certain contributing factors, I do not believe they have provided compelling data on the key regulators of exercise-induced intestinal injury, at least experimentally they did not perform exhaustive experiments to identify such. Nor did the authors include data showing any kind of reconstruction that occurs in the GI after exercise. I believe the authors need to revise this statement to reflect that they investigated certain or specific regulators of the damage response in the intestines after exercise training.

(5) Was water intake monitored and recorded per group? If so I think it would be important to include in the supplemental data. Fluid intake/proper hydration can also contribute to changes in the microbiome and if the data is available, it would complement the food intake. If for any reason the exercise groups were taking in less fluid it may be a confounding factor that should be considered.

(6) Methods section - Treadmill running exercise protocol, line 143, I think there is a typo with "exercise straining". Did the authors mean to write "exercise training"? If it is indeed a typo, the same appears in the supplemental material under the same section.

(7) The microbiome analysis is sufficient, and the authors speculate on the possible consequences of the observed changes to the microbiota. However, I believe Figures 5E-G are misleading. The positive correlation is present because of the increase in gut leakiness and the observed exercise-induced increase in microbes. However the same correlation could be made with any positive adaptation to exercise and the observed gut leakiness. I believe those correlations, as described now, postulate these microbes (members of the family Lachnospiraceae) are associated with increased gut leakiness. However, this correlation is not compelling as it is, and additional experiments are warranted to justify this. It cannot be ruled out that the microbes are increasing due to exercise itself. Additionally, reports have suggested species within the Lachnospiraceae family do increase in response to exercise in mice and are associated with positive adaptations to exercise (PMID: 28862530, PMID: 37940330, PMID: 36517598). With this, it should be noted that Lachnospiraceae was also found to be negatively associated with endurance performance (PMID: 35002754). Therefore, specific species or stains of Lachnospiraceae may be highly responsive to exercise while others are not. Without deeper sequencing it is impossible to tease this out and therefore, the authors should be careful with any interpretation beyond discussing what is observed. Additionally, these correlations between Lachnospiraceae and gut leakiness should be interpreted cautiously or more experiments should be included which demonstrate these microbes are connected to gut leakiness. Much more research is needed to determine exactly what strains are positively and negatively associated with exercise adaptations and performance.

Author response:

We plan to provide full author responses and submit a revised version of our manuscript at the earliest opportunity.