Effects of Voluntary Exercise and Acetic Acid Supplementation on Skeletal Muscle Mitochondrial Function in Ovariectomized Mice

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript presents very interesting data on the effects of exercise and acetate supplementation on mitochondrial function in skeletal muscle in OVX mice.

The Introduction should be more concise, focusing only on the essential background information necessary for the study's objectives.

The possibility that lower exercise levels in the OVX group influenced these results is thoroughly discussed in the Discussion section.

Performing the same interventions in the Sham group as in the OVX group could have revealed whether acetate and exercise are more effective under conditions of estrogen deficiency, such as increased body weight, body fat mass, and oxidative stress. Please add a discussion on this point.

This study investigated the effects of acetic acid and exercise on postmenopausal body composition and skeletal muscle metabolic function. While acetic acid and exercise are thought to contribute to improved metabolic function via mitochondrial function, the study was original in examining the combined effects of both.

Exercise alone did not result in significant improvements in mitochondrial function. As the authors state in their conclusion, it is regrettable that this was not conducted as a forced exercise test.

It is also regrettable that no intervention with acetic acid or exercise was performed in the SHM group. Considering the potential effects of acetic acid or exercise on the SHM group could have influenced the results of this study.

Did the mouse feed contain isoflavones such as genistein or daidzein?

The Discussion section mentions differences in muscle fibers and antioxidant activity between the gastrocnemius and soleus muscles, suggesting these differences may have influenced the study results. Does this mean the increased oxidative stress after ovariectomy was counteracted by the soleus muscle's higher antioxidant capacity?

Author Response

Comments 1: This manuscript presents very interesting data on the effects of exercise and acetate supplementation on mitochondrial function in skeletal muscle in OVX mice. The Introduction should be more concise, focusing only on the essential background information necessary for the study's objectives.

• Thanks a lot for your great comments. Introduction has been revised more concise.

 

Comments 2: The possibility that lower exercise levels in the OVX group influenced these results is thoroughly discussed in the Discussion section.

• Yes, the lower voluntary physical activity or exercise capacity level in the OVX rodents is now thoroughly discussed in the 3^rd paragraph of the Discussion section.

 

Comments 3: Performing the same interventions in the Sham group as in the OVX group could have revealed whether acetate and exercise are more effective under conditions of estrogen deficiency, such as increased body weight, body fat mass, and oxidative stress. Please add a discussion on this point.

• Yes, we agree on your suggestions. However, the inclusion of the sham groups having the same interventions may increase the number of groups from 5 to 8. Unfortunately, the condition of our laboratory and facilities at the time could not cover too many animals. Now we include that point in the discussion as one of the study limitations.

 

Comments 4: This study investigated the effects of acetic acid and exercise on postmenopausal body composition and skeletal muscle metabolic function. While acetic acid and exercise are thought to contribute to improved metabolic function via mitochondrial function, the study was original in examining the combined effects of both.

• Thanks a lot for your positive comments and your comments are totally right. It has been somewhat documented the beneficial effects of exercise intervention or acetic acid supplementation alone. But, whether acetic acid supplementation during exercise can provide a synergistic effect on skeletal muscle and whole-body metabolism was unknown in estrogen-deficient conditions.

 

Comments 5: Exercise alone did not result in significant improvements in mitochondrial function. As the authors state in their conclusion, it is regrettable that this was not conducted as a forced exercise test.

• The study using a forced exercise test will be the future study in our research group. We included that point in the discussion as one of the study limitations.

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Comments 6: It is also regrettable that no intervention with acetic acid or exercise was performed in the SHM group. Considering the potential effects of acetic acid or exercise on the SHM group could have influenced the results of this study.

• Thank you for your thoughtful suggestion. As we responded to your comments #3, unfortunately, the condition of our laboratory and facilities at the time could not cover too many animals. We included your point in the discussion as one of the study limitations.

 

Comments 7: Did the mouse feed contain isoflavones such as genistein or daidzein?

• We used the standard rodent chow (NIH-41KO; Zeigler Bros, Inc., USA), which does not have any isoflavones. We had checked the ingredients of the chow with the provider.

 

Comments 8: The Discussion section mentions differences in muscle fibers and antioxidant activity between the gastrocnemius and soleus muscles, suggesting these differences may have influenced the study results. Does this mean the increased oxidative stress after ovariectomy was counteracted by the soleus muscle's higher antioxidant capacity?

• Thank you for your careful review. Yes, that was our one of the possibilities of different sensitivity to interventions between soleus and gastrocnemius. Our data showed that no alterations were found in soleus mitochondrial function after OVX or other treatments. We did not compare the antioxidant levels between two muscles in the current study, but lots of previous research showed higher numbers and functions of mitochondria along with mitochondria-related antioxidants (i.e. SODs) in soleus compared to gastrocnemius muscle [Ref#51-53]. However, this was only our assumption, thus more studies are necessary to investigate the underlying mechanism of the different sensitivity to interventions between soleus and gastrocnemius, which was mentioned in the 6th paragraph of the Discussion.

Reviewer 2 Report

Comments and Suggestions for Authors

Park et al have done a nice study on the effects of exercise plus acetic acid on ovariectomized mice. The goal of their study was to determine whether exercise (voluntary wheel running) with the addition of consumption of acetic acid (usually in the form of vinegar for humans) would help preserve insulin sensitivity and mitochondrial function/ROS in menopausal women.  The group got mixed responses. These treatments had no effect on body composition and insulin resistance was changed but not more with the acetic acid.  The acetic acid and running did affect the mitochondrial function in the soleus, a muscle involved in movements like running, but not the gastroc, a muscle involved in movements like lifting, (which makes sense because these mice were doing treadmill running).  From western blotting, they looked at levels of AMPKalpha with phosphorylation and PGC1a and did a western for carboxylated proteins.  They got significant results with the ratio of phosphorylated to nonphos. AMPKalpha, but with nothing else.  This study seems very well done and I have some comments that may be able to have the authors see more differences in the data with the Westerns.

Major Comments:

1. The western blots in Fig. 5 and 6 often were very over-saturated which dulls the differences in the groups. Either rerun or use blots that are not saturated for your data to see if you can see any more differences.
2. Also with Fig 5 and 6, you may be able to see differences if you normalize each blot to the control animal. It appears what you are doing is taking the numbers from each and just averaging them, but if the sham animal is always 1, then you may see differences in treatments when you average.

Minor Comments:

1. In the very first sentence of the introduction, you never mention that you are talking about women, not animals. Please just say you are.
2. Also in that paragraph, when you talk about the NHANES data, that is American women, correct? Please specify.  Seems like a high percentage of overweight and obese.
3. In each figure legend, list the number of repeats. Also make the graphs with the data points visible within the bars.

Author Response

Comments 1: The western blots in Fig. 5 and 6 often were very over-saturated which dulls the differences in the groups. Either rerun or use blots that are not saturated for your data to see if you can see any more differences.

• Thanks a lot for your suggestions on western blot images. Some images looked somewhat bad due to dark bands with background noise. However, when we imaged the bands using the Chemidoc, we did not find any over-saturation marks as our software has the detecting function of it. Following your suggestions, we re-ran the analysis using only blots that are not suspicious to be saturated. Unfortunately, nothing got better regarding the group differences.

 

Comments 2: Also with Fig 5 and 6, you may be able to see differences if you normalize each blot to the control animal. It appears what you are doing is taking the numbers from each and just averaging them, but if the sham animal is always 1, then you may see differences in treatments when you average.

• Thanks again for your suggestions on the normalization method. Actually, IT is the great suggestion and that was the way that our lab frequently have used too. Also sometimes we use the normalization of each blot to the total band average of each blot, which would normalize the blot difference. Unfortunately, nothing was moving toward a positive way regarding the group differences using those different normalization methods including your suggestion. Again, we appreciate your great suggestion.

 

Comments 3: In the very first sentence of the introduction, you never mention that you are talking about women, not animals. Please just say you are.

• Thank you for the comments on introduction. The sentence now has been revised accordingly.

 

Comments 4: Also in that paragraph, when you talk about the NHANES data, that is American women, correct? Please specify. Seems like a high percentage of overweight and obese.

• The research was conducted in the United States. The sentence now has been revised accordingly.

 

Comments 5: In each figure legend, list the number of repeats. Also make the graphs with the data points visible within the bars.

• The figures and legends have been revised accordingly.