Myristic Acid Regulates Triglyceride Production in Bovine Mammary Epithelial Cells through the Ubiquitination Pathway
Round 1
Reviewer 1 Report
The authors investigated an important issue regarding to the regulatory mechanism of myristic acid on milk fat synthesis in cows , hence the dairy milk industry should benefits from this study as well as the dairy farms ,They emphasized that the myristic acid's role in regulating triglyceride synthesis in MAC-T cells and its potential application as a feed additive for cattle, benefitting the dairy industry's milk production efficiency and economic outcomes. Which could be consider as an important finding, But in my opinion there are a few cosmetics changes still needs for more enhancement the manuscript General comments
1- The key words need to rearranged according to alphabetical order, if available more keywords will also better for the manuscript distribution
2- The authors should rewrite the hypothesize in the introduction section with clear statement before the objectives please
3- The abbreviations should be emphasized the whole name when first mentioned, please
4- Line 41 ( The bovine mammary gland epithelial cell line MAC-T originated from the frozen stock of our laboratory) Please more information needs to be provide about the MAC-T.
5- Some detergents and media like ( DMEM culture medium, fetal bovine serum (FBS), and peni cillin-streptomycin were purchased from GIBCO? The authors should put more information about this products , like the full scientific classifications , the company origin , country and state, the purity of the products etc… ( This applies to all instruments, chemicals, or cultures that were used over the course of the experiment, Please
6- line 63 more information needs Myristic acid powder? source and manufacture as I mentioned above
7- Line 70 to centrifugation, what about the conditions ? Please
8- Line 90 proteasome activity assay kit , what about this kit ? manufacture , origin etc…
9- In my opinion some missing information in some techniques used in the experiment , the authors should reconsider this point
10- A group of authors has carried out this experiment at the level of the cell itself, and one of the most important things that has been clarified is that this addition will positively affect the fat content of milk, I mean cow's milk, and I expected that such an experiment would be at the level of the animal itself, and not just the cell level To be an actual application for this product, I wish this will be consider in the authors future plan. In addition the cost and visibility study of using the myristic acid should be taken also into consideration Please
11- The results are clear but when authors mentioned increased or decreased the p value should be written please
12- Discussion section line 266 first statement need reference please
13- I have an question for the authors if the myristic acid a promising product for changing the milk fat for better composition how would you apply it for the animals? Furthermore what about the in vivo dose you will use? Also if animals took myristic acid dose it not inter in the metabolic system and may be change his effect on the animal in vivo ? do you warranty this effect ? such points should be taken into considerations please
14- Line . In conclusion, this is not suitable place for the conclusion, please reconsider that
15- -The conclusion is so long need to shorten, please, just to facilitate the reading process
16- Authors should review the references again and write it according to the journal guidance’s
Author Response
- The key words need to rearranged according to alphabetical order, if available more keywords will also better for the manuscript distribution
Thank you for your valuable comments. We have made the following revision: lipid droplets, Myristic acid; MAC-T Cells; Triglycerides; Ubiquitination pthway.
2. The authors should rewrite the hypothesize in the introduction section with clear statement before the objectives please
Thanks for your valuable comments. We have made the following revision: Hence, the focus of our study revolves around the hypothesis that myristic acid poten-tially influences triglyceride (TG) synthesis through the ubiquitination pathway. Our research strategy entails cultivating MAC-T cells within varying concentrations of myristic acid solution. This approach aims to explore the influence of myristic acid on the production of bovine milk fat TG, as well as its potential correlation with the ubiq-uitination signaling pathway.Furthermore, our objective extends to delving into the intricate molecular mechanisms by which myristic acid exercises regulation over milk fat synthesis, specifically through its interaction with the ubiquitination pathway. This comprehensive investigation aims to provide deeper insights into myristic acid's role in the generation of triglycerides within mammary epithelial cells. Additionally, we aspire to discern its viability as a potential feed additive that could enhance milk fat content in cattle.
The abbreviations should be emphasized the whole name when first mentioned, please
Thanks for your valuable comments. We have add the abbreviations of the word when first mentioned.
Line 41 (The bovine mammary gland epithelial cell line MAC-T originated from the frozen stock of our laboratory) Please more information needs to be provide about the MAC-T.
Thanks for your valuable comments. We have made the following revision: The immortalized bovine mammary gland epithelial cell lines MAC-T cells, 40 generations, were provided by the laboratory of Chinese Academy of Sciences, Kunming Institute of Zoology.
5. Some detergents and media like (DMEM culture medium, fetal bovine serum (FBS), and penicillin-streptomycin were purchased from GIBCO? The authors should put more information about this products, like the full scientific classifications, the company origin , country and state, the purity of the products etc (This applies to all instruments, chemicals, or cultures that were used over the course of the experiment, Please.
Thanks for your valuable comments. We have made the following revision: The high glucose Dulbecco’s modified Eagle’s medium (DMED, Gibco™ 11965092, 99%), fetal bovine serum (FBS, Gibco™ 10099-141, 98%), and penicillin-streptomycin (Gibco™ 15140122, concentrated: 100×) were purchased from Thermo Fisher Scientific (United States). The CD36 antibody (No. ab252922) and ADFP antibody (No. a181452) were acquired from Abcam (United States). The ubiquitin antibody (Ubiquitin, sc53509) was purchased from Santa Cruz (United States). The Beta-actin antibody (66009-Ig) was obtained from Protein tech (United States). The HRS-conjugated secondary antibody (7076S) was purchased from Cell Signaling Technology (United States). The myristic acid powder (CAS: 54463-8, 99%) was acquired from Thermo Fisher Scientific (United States). The proteasome activity assay kit (Proteasome-GloTM cell-based Assays, G1180) was purchased from Promega (United States).
6. line 63 more information needs Myristic acid powder? source and manufacture as I mentioned above
Thanks for your valuable comments. The myristic acid powder (CAS: 54463-8, 99%) was acquired from Thermo Fisher Scientific (United States).- Line 70 to centrifugation, what about the conditions ? Please
Thanks for your valuable comments. We have made the following revision: centrifugation (1000rpm, 5 minutes)
8. Line 90 proteasome activity assay kit , what about this kit ? manufacture , origin etc…
Thanks for your valuable comments. We have made the following revision: Proteasome activity was measured in MAC-T cells cultured with different con-centrations of myristic acid using a proteasome activity assay kit, which included ac-tivities of trypsin-like, chymotrypsin-like, and caspase-like proteasomes. The cell sus-pensions from each group were added to a 96-well plate. Prepare the Pro-teasome-Glo™ Cell-Based Reagent and allow the Reagent to equilibrate to room tem-perature. Remove the 96-well plate containing cells from the incubator, and allow the plate to equilibrate to room temperature. Add 100μl of Proteasome-Glo™ Cell-Based Reagent to each 100μl of sample and control. Cover the plate using a plate lid. Mix the contents of the wells at 700rpm using a plate shaker for 2 minutes. Incubate at room temperature for 10 minutes. and mixed with the prepared equal volume of a fluoro-genic proteasome detection reagent. After incubation at room temperature for 10 minutes, tThe relative fluorescence signal value was measured to analyze the pro-teasome activity of the three types of proteasomes in different cell groups.
9. In my opinion some missing information in some techniques used in the experiment , the authors should reconsider this point
Thank you for your insightful comments. We have incorporated the suggested revisions into the manuscript.
10. A group of authors has carried out this experiment at the level of the cell itself, and one of the most important things that has been clarified is that this addition will positively affect the fat content of milk, I mean cow's milk, and I expected that such an experiment would be at the level of the animal itself, and not just the cell level To be an actual application for this product, I wish this will be consider in the authors future plan. In addition the cost and visibility study of using the myristic acid should be taken also into consideration Please
Thank you for your perceptive feedback. We have indeed taken into consideration the potential application of myristic acid in animals. Currently, we are actively engaged in conducting experiments involving both mice and cows to further explore its effects.
11. The results are clear but when authors mentioned increased or decreased the p value should be written please
Thanks for your valuable comments. The suggested revisions have been incorporated into our manuscript. We used T-test to re-analyze the data differences of each group and updated the change-fold and p-values. - Discussion section line 266 first statement need reference please
Thanks for your valuable comments. The suggested revisions have been incorporated into our manuscript, as following: 3.4. Ogunnaike M, Wang H, Zempleni J: Bovine mammary alveolar MAC-T cells afford a tool for studies of bovine milk exo-somes in drug delivery. International journal of pharmaceutics 2021, December 15; 610: 121263.
13. I have an question for the authors if the myristic acid a promising product for changing the milk fat for better composition how would you apply it for the animals? Furthermore what about the in vivo dose you will use? Also if animals took myristic acid dose it not inter in the metabolic system and may be change his effect on the animal in vivo ? do you warranty this effect ? such points should be taken into considerations please
Thank you for your insightful input. We have thoroughly considered the possible implementation of myristic acid in animals. At present, we are deeply immersed in experimental investigations encompassing both mice and cows, with the aim of delving deeper into its potential impacts. Our approach involves meticulously designed studies that encompass controlled parameters and varying dosages of myristic acid. These efforts allow us to closely monitor its influence on milk fat composition and the general well-being of the animals. The stringent design of these studies ensures that any observed alterations can be confidently attributed to the introduction of myristic acid. - Line . In conclusion, this is not suitable place for the conclusion, please reconsider that
I appreciate your valuable feedback. We have incorporated your insights into the conclusion of our manuscript. - The conclusion is so long need to shorten, please, just to facilitate the reading process
I appreciate your valuable feedback. We have incorporated your insights into the conclusion of our manuscript, as following: This study aimed to evaluate how myristic acid influences triglyceride synthesis in bo-vine milk fat. We cultured MAC-T cells with varying myristic acid concentrations and observed that 200μM myristic acid notably increased protein expressions of CD36, ADFP, and UB. Myristic acid didn't disrupt proteasome activity, enhancing cell viabil-ity and indicating minimal cellular stress. Subsequent tests on triglyceride content and lipid droplets confirmed the substantial role of 200μM myristic acid in triglyceride synthesis and lipid content within MAC-T cells. This underscores the safe nature of myristic acid and its dose-dependent effect on triglyceride synthesis. These findings provide insights into myristic acid's cellular-level contribution to milk fat regulation, awaiting validation at the organismal level. The study significantly bolsters our un-derstanding of myristic acid's mechanisms and potential in governing milk fat produc-tion.
16. Authors should review the references again and write it according to the journal guidance’s
Thanks for your valuable comments. We have revised the references according to the journal guidance.
Reviewer 2 Report
Dear authors, it seems to me that this is a good manuscript and it has relevance in the scientific world. However, major technical and/or interpretational problems affect the quality of the manuscript.
Dear authors, first of all, please do not reply to me without proofreading the manuscript if necessary. If I question the topics, it is because the text needs improvement; then change the text if necessary.
Abstract: Complete the summary by adding the statistical design and treatments.
Keywords: It is recommended that the keywords are different from the words used in the title to increase the visibility of the manuscript in future scientific research. In this sense, add and/or change keywords.
Introduction: Add in the introduction in detail what was the scientific basis (chemical, biochemical, physiological, etc.) to believe that myristic acid has a potential role in the synthesis of bovine milk fat if the pathways from the intestine to the gland mammary are many. It is important to note that the changes in these pathways that milk fat can occur at various places on these pathways.
Also add the hypothesis.
Add insights from previous studies that support your theories or hypotheses.
Please add why you choose to assess the protein expression levels of CD36, ADFP and UB, and other metabolites. What is the connection of these metabolites with the production of milk fat?
Line 20: Are you sure? Decanoic acid has 10 carbons and myristic acid has 14. I think there was a typo.
Lines 23-24: Remove “…conducted by our research team” because if it is your previous study, it is natural to understand that it was done by your research team.
Lines 28-29: Add the percentage of myristic acid that is produced from de novo synthesis and from long chain fatty acids. Are you sure that the synthesis of 14-carbon fatty acids from long-chain fatty acids is significant?
Line 34: Remove “additionally”
Line 40: Correct.
Line 65: Exist in figures a CT treatment. Describe this in this part.
Lines 133-134: Why T-tests and no regression (polynomial) tests?
Lines 139-140: This text is not necessary because it is repetitive. Remove it.
Line 142: Dear author, I know and you know what 2.13-fold, 1.71-fold, etc. means. However, it is necessary for the reading to be fluent for new readers as well. Try to detail somewhere in the manuscript what it is –fold, CD36, Ubiquitination, etc.
Lines 146-147: Given these results, I recommend change to regression (polynomial) statistical analysis and understanding whether myristic acid promotes linear or quadratic effects.
Lines 162-163: protein stability from where? Whose?
Figure 4: Improve the figure. Increase the size. Or add this figure as an annex in better resolution and size.
Discussion: Dear authors, your discussion is well written and good to read, congratulations.
Line 228-230: This is not a myristic fatty acid specific pathway. Other molecules can activate this pathway. Describe and add a number to support this statement (Only as an example: in a multivariate analysis, myristic fatty acid showed 83% implicance in fatty acid synthesis)
Lines 291-301: These are a compilation of results. You must describe/discuss biologically how you arrived at these results.
Lines 309-312: I understand your conclusion; however, think with me: if I add glucose to the liver cells; as a result, I will probably be able to see higher glycolysis. If I increase the concentration of any medium or long chain fatty acid (oleic, stearic, linoleic, linolenic, etc.) in the mammary cell, shouldn't an increase in triglyceride synthesis be expected?
My comment focuses on the fact that you need to support your conclusion in an approach that explains your result. A description or summary of the results is not sufficient to support your conclusion.
Minor editing of English language required
Author Response
Abstract: Complete the summary by adding the statistical design and treatments.
Thank you for your valuable comments. We have made the following revision: This study investigated the regulatory mechanism of myristic acid on milk fat synthesis in cows. The association between myristic acid and high milk fat content in Zhongdian yaks' gut was found through combined metagenomics and metabolomics analysis. Bovine mammary epithelial cells (MAC-T) were cultured and treated with various myristic acid concentrations. After 24 hours, protein expression levels of CD36 (Membrane Glycoprotein CD36), ADFP (Adipose Differ-entiation-Related Protein ADFP), and UB (Ubiquintin) were analyzed, along with cellular proteasome activity, triglyceride content, lipid droplets and cell viability. Myristic acid at 200μM significantly upregulated CD36, ADFP, UB, and the content of triglyceride and lipid droplets, and cell viability, but did not affect proteasome activity. Pathway analysis revealed that myristic acid regulates milk fat synthesis through ubiquitination-lysosome and ubiquitination-proteasome pathways. The study demonstrates myristic acid's role in regulating triglyceride synthesis in MAC-T cells and its potential application as a feed additive for cattle, benefitting the dairy industry's milk production efficiency and economic outcomes.
Keywords: It is recommended that the keywords are different from the words used in the title to increase the visibility of the manuscript in future scientific research. In this sense, add and/or change keywords.
Thank you for your valuable comments. We have made the following revision: lipid droplets, Myristic acid; MAC-T Cells; Triglycerides; Ubiquitination pathway
Introduction: Add in the introduction in detail what was the scientific basis (chemical, biochemical, physiological, etc.) to believe that myristic acid has a potential role in the synthesis of bovine milk fat if the pathways from the intestine to the gland mammary are many. It is important to note that the changes in these pathways that milk fat can occur at various places on these pathways.
Thank you for your invaluable feedback. We have extensively discussed the potential role of myristic acid in milk fat synthesis in the "Discussion" section. To ensure conciseness and avoid repetition, the introduction now solely presents the underlying assumptions.
Also add the hypothesis.
Thanks for your valuable comments. We have made the following revision: Hence, the focus of our study revolves around the hypothesis that myristic acid poten-tially influences triglyceride (TG) synthesis through the ubiquitination pathway. Our research strategy entails cultivating MAC-T cells within varying concentrations of myristic acid solution. This approach aims to explore the influence of myristic acid on the production of bovine milk fat TG, as well as its potential correlation with the ubiquitination signaling pathway.Furthermore, our objective extends to delving into the intricate molecular mechanisms by which myristic acid exercises regulation over milk fat synthesis, specifically through its interaction with the ubiquitination pathway. This comprehensive investigation aims to provide deeper insights into myristic acid's role in the generation of triglycerides within mammary epithelial cells. Additionally, we aspire to discern its viability as a potential feed additive that could enhance milk fat content in cattle.
Add insights from previous studies that support your theories or hypotheses.
Thank you for your insightful feedback. We have implemented the suggested revisions as indicated earlier and in discussion. And also incorporated your insights into the introduction, as following: The primary component of milk at is triglyceride (TG), which is synthesized mainly by mammary epithelial cells either by de novo synthesis from precursor mole-cules like acetate or by direct utilization of long-chain fatty acids present in the blood [3]. Myristic acid, a 14-carbon fatty acid, may could be directly employed in the syn-thesis of triglycerides. Myristic acid, a 14-carbon fatty acid, could potentially be uti-lized directly in triglyceride synthesis, although there is limited existing research on this topic.
Please add why you choose to assess the protein expression levels of CD36, ADFP and UB, and other metabolites. What is the connection of these metabolites with the production of milk fat?
We value your feedback, and I'd like to provide further information. CD36 acts as a transport protein located on the membrane of MAC-T cells. Its role involves the absorption of long-chain fatty acids, contributing directly to the regulation of lipid intake. Specifically, CD36 is capable of transporting myristic acid, a 14-carbon fatty acid. Another significant component, ADFP, serves as a marker protein for lipid droplets. It aids in evaluating both the triglyceride content and the presence of lipid droplets within the cells.
In relation to UB, it represents the overall level of ubiquitinated proteins within the cell. This measurement serves as an indicator of the ubiquitination accumulation occurring in MAC-T cells. Notably, prior research has established a direct link between the ubiquitination pathway and the regulation of CD36 and ADFP. Given these insights, our focus lies on assessing these specific proteins due to their integral roles in the context mentioned.
Line 20: Are you sure? Decanoic acid has 10 carbons and myristic acid has 14. I think there was a typo.
We appreciate your valuable feedback. We have revised it in the introduction of our manuscript.
Lines 23-24: Remove “…conducted by our research team” because if it is your previous study, it is natural to understand that it was done by your research team.
I appreciate your valuable feedback. We have removed it in our manuscript.
Lines 28-29: Add the percentage of myristic acid that is produced from de novo synthesis and from long chain fatty acids. Are you sure that the synthesis of 14-carbon fatty acids from long-chain fatty acids is significant?
Thanks for your insightful feedback. In this section, our objective is to expound on the synthesis of triglycerides, drawing insights from existing literature. This synthesis can occur via de novo processes involving small molecules like acetate (predominant in the later stages of cow lactation), or directly through the synthesis of medium and long-chain fatty acids (more prominent in the early lactation phases of cows). Notably, myristic acid, a 14-carbon fatty acid, can be directly employed in the synthesis of triglycerides. Regarding the percentage of myristic acid originating from de novo synthesis and from long-chain fatty acids, we currently lack this information and it is not within the scope of our present study. We acknowledge your valuable input and intend to incorporate your suggestions for further research in our upcoming studies.
Line 34: Remove “additionally”
We appreciate your valuable feedback. We have removed it in our manuscript.
Line 40: Correct.
We appreciate your valuable feedback. We have revised it in our manuscript as following: Reagents.
Line 65: Exist in figures a CT treatment. Describe this in this part.
Thank you for your insightful feedback. We have incorporated your suggestions into our manuscript, resulting in the following revision: In contrast, the control group (CT) was cultured in complete DMEM medium supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin, without the presence of myristic acid.
Lines 133-134: Why T-tests and no regression (polynomial) tests?
Thank you for your valuable input. In our initial draft, we carried out an analysis of variance among distinct groups; however, in the figures, we solely focused on contrasting differences between different concentration groups and the control group. Consequently, we decided to reemploy the T-test and One-way ANOVA for conducting a comparative analysis and also made revisions to the figures.
Regarding the regression analysis, we applied regression techniques to our preliminary metabolomic data, revealing a significant correlation and noteworthy regression connection between myristic acid and a high milk fat percentage within a multitude of metabolites. This study was conducted based on the well-established positive correlation observed between myristic acid and an increased milk fat percentage.
Moreover, this study encompassed just four concentration groups: CT, 100μM, 150μM, and 200μM, each comprising only 3-4 replicates. Consequently, accurately calculating the regression between myristic acid concentration and protein expression or TG content was not feasible. We are currently considering the possibility of conducting subsequent experiments involving mice and cows.
Lines 139-140: This text is not necessary because it is repetitive. Remove it.
We appreciate your valuable feedback. We have removed it in our manuscript.
Line 142: Dear author, I know and you know what 2.13-fold, 1.71-fold, etc. means. However, it is necessary for the reading to be fluent for new readers as well. Try to detail somewhere in the manuscript what it is –fold, CD36, Ubiquitination, etc.
Thank you for your insightful feedback. We have incorporated your suggestions into our manuscript, and have add the abbreviations of the word when first mentioned.
Lines 146-147: Given these results, I recommend change to regression (polynomial) statistical analysis and understanding whether myristic acid promotes linear or quadratic effects.
Thank you for your valuable input. In our initial draft, we carried out an analysis of variance among distinct groups; however, in the figures, we solely focused on contrasting differences between different concentration groups and the control group. Consequently, we decided to reemploy the T-test and One-way ANOVA for conducting a comparative analysis and also made revisions to the figures.
Regarding the regression analysis, we applied regression techniques to our preliminary metabolomic data, revealing a significant correlation and noteworthy regression connection between myristic acid and a high milk fat percentage within a multitude of metabolites. This study was conducted based on the well-established positive correlation observed between myristic acid and an increased milk fat percentage.
Moreover, this study encompassed just four concentration groups: CT, 100μM, 150μM, and 200μM, each comprising only 3-4 replicates. Consequently, accurately calculating the regression between myristic acid concentration and protein expression or TG content was not feasible. We are currently considering the possibility of conducting subsequent experiments involving mice and cows.
Lines 162-163: protein stability from where? Whose?
Thank you for your insightful feedback. We have incorporated your suggestions into our manuscript, and made revision as following: This suggests that the three concentrations of myristic acid may alter the protein ubiquitination level in MAC-T cells but do not affect protein homeostasis maintained by proteasome activity.
Figure 4: Improve the figure. Increase the size. Or add this figure as an annex in better resolution and size.
Thank you for your insightful feedback. We have incorporated your suggestions into our manuscript, and improved the resolution and size of figure 4.
Discussion: Dear authors, your discussion is well written and good to read, congratulations.
Your affirmation is truly valued and acts as a significant source of encouragement for us. We sincerely thank you once more for your kind words!
Line 228-230: This is not a myristic fatty acid specific pathway. Other molecules can activate this pathway. Describe and add a number to support this statement (Only as an example: in a multivariate analysis, myristic fatty acid showed 83% implicance in fatty acid synthesis)
Thank you for your valuable insights. Regarding the correlation between myristic acid and fatty acid synthesis, most current research only establishes a certain level of regulatory relationship between the two, without definitive data indicating the extent of their correlation. In our preliminary data on metabolites from the intestines of Zhongdian yaks, we have observed a certain degree of positive correlation between myristic acid and high milk fat content (as shown in the figures below). However, the contribution of myristic acid to the process of fatty acid synthesis has not been definitively determined.
Lines 291-301: These are a compilation of results. You must describe/discuss biologically how you arrived at these results.
Thanks for your valuable comments. We have incorporated your insights into the discussion of our manuscript, as following: The outcomes unveiled a significant correlation: the exposure of MAC-T cells to a concentration of 200μM myristic acid yielded a substantial augmentation in the ex-pression level of ADFP. Positioned on the surface of lipid droplets within cells, ADFP assumes a pivotal role in fostering the creation of intracellular lipid droplets [25-26]. Notably, existing research, albeit unpublished, has pointed out that ADFP possesses the capability to directly interact with ubiquitin proteins, governed by the ubiquitina-tion-proteasome signaling pathway. The noticeable surge in ADFP expression effec-tively signifies a concurrent increase in the count of lipid droplets housed within the cells. Moreover, our investigation brought to light a compelling finding: the infusion of 200μM myristic acid notably amplified the triglyceride content within MAC-T cells. This conclusion was fortified by the utilization of Nile red staining, which revealed a conspicuous augmentation in the lipid droplet content within the cells. This concur-rence between the two observations serves to reinforce the hypothesis that myristic acid significantly stimulates triglyceride synthesis within MAC-T cells. As a cumula-tive result, this process ultimately propels the production of milk fat.
To summarize, our results lay a biological foundation that connects the elevation in ADFP expression, the surge in lipid droplet count, and the augmented triglyceride content to the administration of 200μM myristic acid. This interplay underscores myristic acid's role in bolstering intracellular lipid accumulation, subsequently con-tributing to the enhancement of milk fat production in MAC-T cells.
Lines 309-312: I understand your conclusion; however, think with me: if I add glucose to the liver cells; as a result, I will probably be able to see higher glycolysis. If I increase the concentration of any medium or long chain fatty acid (oleic, stearic, linoleic, linolenic, etc.) in the mammary cell, shouldn't an increase in triglyceride synthesis be expected?
We appreciate your valuable feedback, and your reasoning is generally correct. Increasing the concentration of medium or long-chain fatty acids in mammary cells would likely result in an increase in triglyceride synthesis. These fatty acids serve as the building blocks for triglycerides, which are the primary storage form of energy in adipose tissue and also play a role in milk production in mammary cells. When fatty acids are abundant, mammary cells can take up these fatty acids and convert them into triglycerides through a process known as lipogenesis. These triglycerides can then be stored within the mammary cells or used to produce milk fat. However, the regulatory mechanisms of these substances on milk fat synthesis and their potential toxicity to mammary epithelial cells or mammary tissue require rigorous validation. In our subsequent research, we will further validate the regulatory effects of additional substances on milk fat synthesis at the cellular or animal level.
My comment focuses on the fact that you need to support your conclusion in an approach that explains your result. A description or summary of the results is not sufficient to support your conclusion.
We appreciate your valuable feedback. We have incorporated your insights into the conclusion of our manuscript, as following: This study aimed to evaluate how myristic acid influences triglyceride synthesis in bo-vine milk fat. We cultured MAC-T cells with varying myristic acid concentrations and observed that 200μM myristic acid notably increased protein expressions of CD36, ADFP, and UB. Myristic acid didn't disrupt proteasome activity, enhancing cell viabil-ity and indicating minimal cellular stress. Subsequent tests on triglyceride content and lipid droplets confirmed the substantial role of 200μM myristic acid in triglyceride synthesis and lipid content within MAC-T cells. This underscores the safe nature of myristic acid and its dose-dependent effect on triglyceride synthesis. These findings provide insights into myristic acid's cellular-level contribution to milk fat regulation, awaiting validation at the organismal level. The study significantly bolsters our un-derstanding of myristic acid's mechanisms and potential in governing milk fat produc-tion.
Round 2
Reviewer 2 Report
Dear authors, it seems to me that this is a good manuscript and it has relevance in the scientific world. In this sense, I recommend aproving the manuscript.
Author Response
Thank you for your valuable insights. And your affirmation is truly valued and acts as a significant source of encouragement for us. We sincerely thank you once more for your kind words!
