Mitochondrial DNA copy number, metabolic syndrome, and insulin sensitivity: Insights from the Sugar, Hypertension, and Physical Exercise studies

Stephanie Y. Yang; Caleb S. Mirabal; Charles E. Newcomb; Kerry J. Stewart; Dan E. Arking

doi:10.1371/journal.pone.0270951

Peer Review History

Original SubmissionNovember 23, 2021
12 May 2022 Decision Letter - Victoria J. Vieira-Potter, Editor PONE-D-21-37204Mitochondrial DNA copy number and insulin sensitivity: Insights from the Sugar, Hypertension, and Physical Exercise StudiesPLOS ONE Dear Dr. Arking, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Jun 26 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access. We will update your Data Availability statement to reflect the information you provide in your cover letter. Additional Editor Comments: Due to this unfortunate circumstance of of only one peer review, I am making the decision "major revision". Please refer to the reviewer's comments. The decision was reject, but I do not feel comfortable rejecting the paper outright based on only one review. Hopefully these comments will be helpful as you revise your work either to resubmit to PLOS one or elsewhere. Best Regards, VVP [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't Know ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: This study capitalized on a rich human dataset, including V02 max data and pre and post exercise-intervention data including several metabolic variables on obese individuals. This study correlates these data with circulating levels of mtDNA-CN, an emerging indicator of metabolic health. Although interesting, the new data provided (above what has already been published in the SHAPE interventions) do not seem to warrant publication on their own. Most of the data are not significant, yet some are confirmatory, which is somewhat beneficial to the field. It is particularly interesting that there are age and sex differences. The data do not seem to support strongly a relationship between exercise training and mtDNA-CN, but perhaps the lack of relationship was due to the population mostly being obese and not highly fit. The vast majority of data are presented as supplementary figures, indicating that most of the content is not deemed of sufficient value to most readers (by the authors). There are some interesting findings here. It would be of value if the authors did have skeletal muscle biopsy data to relate to the mtDNA-CN data. This would highlight the potential clinical value that mtDNA-CN may have, leading future studies to use blood mtDNA-CN as an indicator of skeletal muscle quality/mitochondrial activity. The finding that baseline values predicted dropout is peculiar and interesting on its own. If the authors could dig deeper into the data to find a potential explanation for this finding, that could be a central novel element of the paper. Unfortunately, as it currently reads, the paper lacks a major novel finding. It appears that the major finding is that exercise training does not affect mtDNA-CN in obese individuals. Maybe this is an important enough finding to warrant publication, but I just was not convinced of this, especially given the study limitations. I offer my suggestions below and hope this comments are helpful to the authors. Introduction: Line 50, Do you mean “positively” correlated? Are there ever instances where mtDNA-CN are elevated in conditions where mitochondrial are dysfunction, such as with dysfunction in mitophagy pathways leading to greater numbers of dysfunctional mitochondria? Line 52, Please describe “buffy coat” for readers unfamiliar with blood analysis Line 58, has shown should be have shown Line 59, for should be of? More detailed description of “exercise training” is required. Type of exercise? Subject population? Duration/chronicity/intensity? Results: The sex differences are interesting and important. How does age affect the sex difference? Have any studies addressed how estrogen or ot her sex hormones may affect mtDNA-CN? The “effect estimates” that are described – are there p values associated with these graphs (Supp Fig 3)? The dropout/baseline mtDNA-CN values: a more detailed explanation of what potential covariates were ruled out to explain this relationship would be helpful. (eg, age? BMI? Some sociological variable?) ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0270951.r001
Revision 1
16 Jun 2022 Author Response We thank the reviewer for their comments, and have responded to them below. Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: This study capitalized on a rich human dataset, including V02 max data and pre and post exercise-intervention data including several metabolic variables on obese individuals. This study correlates these data with circulating levels of mtDNA-CN, an emerging indicator of metabolic health. Although interesting, the new data provided (above what has already been published in the SHAPE interventions) do not seem to warrant publication on their own. Most of the data are not significant, yet some are confirmatory, which is somewhat beneficial to the field. It is particularly interesting that there are age and sex differences. The data do not seem to support strongly a relationship between exercise training and mtDNA-CN, but perhaps the lack of relationship was due to the population mostly being obese and not highly fit. The vast majority of data are presented as supplementary figures, indicating that most of the content is not deemed of sufficient value to most readers (by the authors). There are some interesting findings here. It would be of value if the authors did have skeletal muscle biopsy data to relate to the mtDNA-CN data. This would highlight the potential clinical value that mtDNA-CN may have, leading future studies to use blood mtDNA-CN as an indicator of skeletal muscle quality/mitochondrial activity. The finding that baseline values predicted dropout is peculiar and interesting on its own. If the authors could dig deeper into the data to find a potential explanation for this finding, that could be a central novel element of the paper. Unfortunately, as it currently reads, the paper lacks a major novel finding. It appears that the major finding is that exercise training does not affect mtDNA-CN in obese individuals. Maybe this is an important enough finding to warrant publication, but I just was not convinced of this, especially given the study limitations. I offer my suggestions below and hope this comments are helpful to the authors. We thank the reviewer for their insightful comments and suggestions. While we agree with the reviewer’s conclusions that the current data does not support a relationship between exercise training and mtDNA-CN, we would like to note the criteria for publication in PLoS One (taken directly from the website) are as follows: 1. The study presents the results of original research. 2. Results reported have not been published elsewhere. 3. Experiments, statistics, and other analyses are performed to a high technical standard and are described in sufficient detail. 4. Conclusions are presented in an appropriate fashion and are supported by the data. 5. The article is presented in an intelligible fashion and is written in standard English. 6. The research meets all applicable standards for the ethics of experimentation and research integrity. We believe that the current study and all analyses were performed with scientific rigor, and that all conclusions in the paper are supported by the data. Furthermore, the manuscript is written in standard English, has not been published elsewhere, and describes the results of original research. Research was conducted in accordance with IRB protocols (IRB00071780), and all clinical studies are listed on ClinicalTrials.gov (SHAPE3: NCT00928005, SHAPE5: NCT00990457). Given this, we believe that this manuscript meets all the criteria for publication in PLoS One. Though negative results are not as exciting or flashy, they are still important and should be disseminated to the rest of the research community. Introduction: Line 50, Do you mean “positively” correlated? Are there ever instances where mtDNA-CN are elevated in conditions where mitochondrial are dysfunction, such as with dysfunction in mitophagy pathways leading to greater numbers of dysfunctional mitochondria? We thank the reviewer for this suggestion and have clarified and adjusted the text on line 50 to read as follows: Higher mtDNA-CN levels are positively associated with mitochondrial membrane potential, respiratory enzyme function, and energy reserves [1,2]… There have been instances where elevated mtDNA-CN is associated with cancer, however, we have not seen literature where elevated mtDNA-CN is associated with mitochondrial dysfunction. Line 52, Please describe “buffy coat” for readers unfamiliar with blood analysis We have adjusted the text on lines 52-53 to define buffy coat: Lower mtDNA-CN in buffy coat, the fraction of blood that contains leukocytes and platelets, has been associated with frailty, often characterized by decreased muscle tone [3]. Line 58, has shown should be have shown We disagree with this suggestion, as we believe version 1 of the sentence is more grammatically sound than version 2. [1] Indeed, persistent exercise training has been shown to increase mitochondrial function and mitochondrial volume in skeletal muscle [4,5]. [2] Indeed, persistent exercise training have shown to increase mitochondrial function and mitochondrial volume in skeletal muscle [4,5]. Line 59, for should be of? We have corrected this sentence. More detailed description of “exercise training” is required. Type of exercise? Subject population? Duration/chronicity/intensity? Later on in the Methods section of the paper, we rigorously describe the type of exercise (lines 271-291), the subject populations (lines 262-269), and the duration/chronicity/intensity (lines 271-291). We agree with the reviewer that increased detail in the introduction would be useful, and have included some more details in that section (lines 61-64): To answer this question, we used two randomized controlled exercise intervention cohorts. Participants were aged 30-65 years, and performed aerobic and resistance exercise training 3 times a week for a duration of 6 months. We have included the more detailed methods sections below for ease of reference: SHAPE3 Subjects were overweight or obese (BMI between 26 and 42 kg/m2), sedentary men and women (n=77), 30-65 years, with prediabetes or diabetes, according to American Diabetes Association criteria (fasting glucose > 126 mg/dl, casual plasma glucose > 200 mg/dl, or 2-hour plasma glucose > 200 mg/dl after a 75-gram oral glucose load). Individuals with uncontrolled diabetes, defined as fasting blood glucose over 300 mg/dl or A1C > 11% were excluded. SHAPE5 Subjects were overweight or obese, sedentary men and women (n=77), BMI 25-42 kg/m2, 30-65 years, who were otherwise healthy. Exercise and Diet Intervention SHAPE3 Diet intervention for SHAPE3 was a nutritionally balanced, moderately hypocaloric diet with reduced saturated fat consistent with American Diabetes Association guidelines. The diet was adjusted to produce a 600 kcal deficit/day for each individual, using resting metabolic rate calculated from the Mifflin-St Jeor equation [6]. Exercise intervention for SHAPE3 was designed based on guidelines from the American College of Sports Medicine and the American Diabetes Association, consisting of warm-up, 45 minutes of aerobic exercise, several resistance training exercises, and cool-down. Exercise sessions were supervised by exercise physiologists to ensure safety and that the exercises were carried out properly. Individuals assigned to exercise intervention were asked to exercise 3 times a week over a 26-week period. SHAPE5 The low-carbohydrate (CHO) group adhered to the New Atkins for Life diet, consisting initially of 15% CHO, 30% protein, and 55% fat, followed by a gradual shift to 40% CHO, 20% protein, and 40% fat. The low-fat group followed American Heart Association (AHA) and National Cholesterol Education Program (NCEP) guidelines, following a diet of 30% fat, 50-55% CHO, and 15-20% protein. All subjects in SHAPE5 participated in 3 times per week supervised exercise training following ACSM guidelines for moderate intensity aerobic and resistance training, consisting of 45 minutes of aerobic exercise and 2 sets of 7 resistance exercises. Results: The sex differences are interesting and important. How does age affect the sex difference? Have any studies addressed how estrogen or other sex hormones may affect mtDNA-CN? It has been well-established that mtDNA-CN varies between sexes[7,8]. While we agree with the reviewer that these questions are interesting and important, we believe that they fall beyond the scope of this manuscript, as the goal of this study was to examine the effect of exercise on mtDNA-CN. The “effect estimates” that are described – are there p values associated with these graphs (Supp Fig 3)? We thank the reviewer for this observation, and agree that this information should be included. We have revised the figure to include effect size estimates and p-values. As a spline does not significantly improve the model for mtDNA-CN and age, we have used a simple linear regression for the plots that are stratified by study. The dropout/baseline mtDNA-CN values: a more detailed explanation of what potential covariates were ruled out to explain this relationship would be helpful. (eg, age? BMI? Some sociological variable?) To clarify covariates that were examined, we have added details on the potential underlying covariates that we evaluated for correlation with dropout (lines 118-126), along with a table (Supplemental Table 1). To understand what could be driving the relationship between baseline mtDNA-CN and study completion, we examined associations between study completion and several other variables. However, none of these potential explanatory variables was significantly associated with study completion. We note that increased age was nominally significantly associated with dropout, however, since increased age is associated with lower mtDNA-CN, this would not explain the observed relationship between higher mtDNA-CN and increased rates of study dropout (Supplemental Table 1). After these analyses, we were unable to account for the variation in dropout explained by baseline mtDNA-CN, and currently do not have a biological explanation for this finding. Variable Name Odds Ratio p-value Age 1.055 0.012 BMI 1.018 0.679 Insulin level 1.004 0.734 Sleep score 1.007 0.760 C-reactive protein 1.014 0.734 Supplemental Table 1. Odds ratios and p-values for associations between explanatory variables and study completion. There was no significant relationship between study completion and variables of interest, save for age. However, the directionality of the association between age and study completion does not explain the relationship between higher mtDNA-CN and higher study dropout. References 1. Guha M, Avadhani NG. Mitochondrial retrograde signaling at the crossroads of tumor bioenergetics, genetics and epigenetics. Mitochondrion. 2013;13: 577–591. doi:10.1016/j.mito.2013.08.007 2. Jeng J-Y, Yeh T-S, Lee J-W, Lin S-H, Fong T-H, Hsieh R-H. Maintenance of mitochondrial DNA copy number and expression are essential for preservation of mitochondrial function and cell growth. J Cell Biochem. 2008;103: 347–357. doi:10.1002/jcb.21625 3. Ashar FN, Moes A, Moore AZ, Grove ML, Chaves PHM, Coresh J, et al. Association of Mitochondrial DNA levels with Frailty and All-Cause Mortality. J Mol Med Berl Ger. 2015;93: 177–186. doi:10.1007/s00109-014-1233-3 4. Jacobs RA, Lundby C. Mitochondria express enhanced quality as well as quantity in association with aerobic fitness across recreationally active individuals up to elite athletes. J Appl Physiol. 2013;114: 344–350. doi:10.1152/japplphysiol.01081.2012 5. Menshikova EV, Ritov VB, Fairfull L, Ferrell RE, Kelley DE, Goodpaster BH. Effects of Exercise on Mitochondrial Content and Function in Aging Human Skeletal Muscle. J Gerontol A Biol Sci Med Sci. 2006;61: 534–540. 6. Mifflin MD, St Jeor ST, Hill LA, Scott BJ, Daugherty SA, Koh YO. A new predictive equation for resting energy expenditure in healthy individuals. Am J Clin Nutr. 1990;51: 241–247. doi:10.1093/ajcn/51.2.241 7. Tin A, Grams ME, Ashar FN, Lane JA, Rosenberg AZ, Grove ML, et al. Association between Mitochondrial DNA Copy Number in Peripheral Blood and Incident CKD in the Atherosclerosis Risk in Communities Study. J Am Soc Nephrol JASN. 2016;27: 2467–2473. doi:10.1681/ASN.2015060661 8. Knez J, Winckelmans E, Plusquin M, Thijs L, Cauwenberghs N, Gu Y, et al. Correlates of Peripheral Blood Mitochondrial DNA Content in a General Population. Am J Epidemiol. 2016;183: 138–146. doi:10.1093/aje/kwv175 Attachments Attachment Submitted filename: Response_To_Reviewer.docx https://doi.org/10.1371/journal.pone.0270951.r002
22 Jun 2022 Decision Letter - Hans-Peter Kubis, Editor Mitochondrial DNA copy number, metabolic syndrome, and insulin sensitivity: Insights from the Sugar, Hypertension, and Physical Exercise Studies PONE-D-21-37204R1 Dear Dr. Arking, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Hans-Peter Kubis, PD. Dr. rer. nat. Academic Editor PLOS ONE Additional Editor Comments (optional): Dear Dr Arking, Thank you for the resubmission of your manuscript. The manuscript was reviewed in the current form and is now acceptable for publication in PLOS ONE. We thank you for submitting your work to PLOS ONE and hope to see more manuscripts being sent to us in the future. Many thanks for considering PLOS ONE and good luck for your future research. https://doi.org/10.1371/journal.pone.0270951.r003
Formally Accepted
8 Jul 2022 Acceptance Letter - Hans-Peter Kubis, Editor PONE-D-21-37204R1 Mitochondrial DNA copy number, metabolic syndrome, and insulin sensitivity: Insights from the Sugar, Hypertension, and Physical Exercise Studies Dear Dr. Arking: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Hans-Peter Kubis Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0270951.r004

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