Mark4 Inhibited the Browning of White Adipose Tissue by Promoting Adipocytes Autophagy in Mice
Round 1
Reviewer 1 Report
The manuscript has been modified according to all the reviewer suggestions.
Author Response
Some amendments:
Line 9, we exchanged “increased” to “increases”.
Line 13, we exchanged “. Under these two model conditions, we” to “and”.
Line 14, we added “significantly”.
Line 15, we deleted “significantly” and added “tests”.
Line 17, we deleted “pathway” and added “Moreover,”.
Line 27, we deleted “in”.
Line 32, we exchanged “are” to “were”.
Line 33, we added “research” and “that”.
Line 34, we exchanged “promotes” to “promoted”.
Line 35, we exchanged “triggers” to “triggered”.
Line 36, we exchanged “promotes” to “promoted”.
Line 38, we exchanged “established” to “establish” and added “a”.
Line 39, we exchanged “roles” to “role”.
Line 40, we deleted “or self-eating”.
Line 56, we deleted “Macroautophagy has been widely studied and profound form of autophagy [14,15,16,17,18]” and added “-induced loss of lipid droplets was”.
Line 57, we added “, whereas the” and “increased”
Line 58, we exchanged “19” to “14” and exchanged “inhibited” to “inhibits”.
Line 59, we exchanged “decreased” to “decreases” and exchanged “An” to “The”.
Line 60, we deleted “mouse” and exchanged “generated” to “generates” and exchanged “lean mice with decreased white adipose mass and enhanced” to “enhancement of” and added “the”.
Line 61, we added “mouse” and “, resulting in the lower of white adipose mass”.
Line 62, we exchanged “20” to “15” and added “the” and added “and essential for the” and deleted “during an”.
Line 63, we exchanged “21” to “16”.
Line 64, we exchanged “induces” to “ induced”.
Line 65, we exchanged “22” to “17”.
Line 66, we exchanged “inhibits” to “inhibited”.
Line67, we exchanged “23” to “18”.
Line68, we exchanged “had” to “have a” and exchanged “were” to “been”.
Line 69, we exchanged “was” to “is”
Line 71, we exchanged “24” to “19” and deleted “can” and exchanged “inhibit” to “inhibited”.
Line 73, we exchanged “25” to “20”.
Line 74, we exchanged “between” to “among” and deleted “and”.
Line 75, we exchanged “26,27” to “21,22”.
Line 76, we exchanged “And” to “Furthermore,”.
Line 77, we added “that”.
Line 79, we deleted “providing a theoretical basis for fat metabolism-related diseases,” and exchanged “the” to “a” and added “strategy” and deleted “of Mark4 as a drug” and added “and”.
Line 86, we deleted “of the cells are” and added “were”.
Line 89, we deleted “that of”.
Line 90, we exchanged “is” to “was”.
Line 91, we added “was” and exchanged “associates” to “associated”.
Line 261, we exchanged “concluded” to “conclude”.
Line 283, we exchanged “changes in” to “occurrence of”.
Line 292, we exchanged “but” to “whereas”.
Line 293, we exchanged “higher” to “elevated”.
Line 294, we deleted “that of” and deleted “under serum starvation”.
Line 296, we deleted “expression and”.
Line 297, we deleted “can” and exchanged “affect” to “affects”.
Line 306, we exchanged “was” to “were”.
Line 307, we exchanged “were” to “was”.
Line 309, we exchanged “treatment” to “treating”.
Line 343, we exchanged “confirms” to “confirmed” and exchanged “promotes” to “promoted”.
Line 345, we added “The” and added “that”.
Line 346, we exchanged “that of” to “the”.
Line 348, we deleted “higher”.
Line 350, we deleted “that of”.
Line 352, we exchanged “After” to “By”.
Line 354, we exchanged “after” to “through”.
Line 355, we exchanged “mTOR” to “mTOR-”.
Line 358, we added “, with”.
Line 380, we exchanged “between” to “among”.
Line 382-383, we deleted “on the basis of serum starvation treatment” and added “on the basis of serum starvation treatment with smaller” and deleted “but the” and deleted “became smaller”.
Line 395, we exchanged “After” to “By”.
Line 399, we exchanged “expression” to “expressions”.
Line 401, we exchanged “this” to “these” and exchanged “conclusion” to “findings”.
Line 402, we exchanged “are” to “were”.
Line 403, we added “were”.
Line 429, we added “were”.
Line 432, we exchanged “conclude” to “concluded”.
Line 433, we exchanged “on” to “in”.
Line 447, exchanged “adipocyte” to “adipocytes”.
Line 448, we added “it is”.
Line 452, we added “, which”.
Line 454, we exchanged “28” to “23”.
Line 458, we exchanged “promotes” to “promoted”.
Line 459, we exchanged “And the” to “The”.
Line 460, we exchanged “were” to “are”.
Line 461, we exchanged “established” to “establish”.
Line 462, we exchanged “on” to “in”.
Line 464, we exchanged “16” to “24” and added “the”.
Line 467, we exchanged “17” to “25” and exchanged “speculated” to “speculate”.
Line 471, we deleted “successfully”.
Line 479, we exchanged “29,30” to “26,27”.
Line 481, we exchanged “30,31” to “28,29”.
Line 482, we exchanged “treated” to “treating”.
Line 486, we added “that”.
Line 491, we exchanged “33” to “30”.
Line 492, we added “the”.
Line 493, we exchanged “34,35” to “31,32”.
Line 511, we exchanged “amount” to “number” and exchanged “increased” to “increases” and exchanged “36,37” to “33,34”.
Line 512, we exchanged “seemed” to “seem”.
Line 513, we exchanged “38” to “35”.
Line 514, we exchanged “39” to “36”.
Line 517, we exchanged “is” to “was”.
Line 519, we exchanged “40,41,42” to “37,38,39” and exchanged “And the” to “This”.
Line 569, we exchanged “43” to “40”.
Line 582, we exchanged “25” to “20”.
Reviewer 2 Report
The study by Yang et al investigates the relation of Mark4 on AMPK and autophagy and browning. The authors dominately uses the 3T3-L1 adipocyte culture models and verify some of their findings in obesity mouse models or mice after cold acclimatisation. However, data in vivo are mostly discriptive. In general, my version is a preliminary version with authors comments and deletions, which makes ist hard to evaluate. On top of this, figures labeling is very small, making it even harder to read. However, besides some specific concerns listed below, this study gives some molecular insights into Mark4 involvement in regulation of autophagy, which is interesting.
Specific comments:
- Line 94: the authors state that starvation and Rpa treatment does not affect cell viability, which is in contrast to the data shown in Fig. 1D and 2A.
- To evaluate GFP-positive puncta, the transfection efficiancy and stability is of major importance. Has that been checked?
- Browning efffects should also be studied by induced browing of 3T3-L1 cells upon beta-receptor stimulation, which is more relvant to the in vivo situation.
- Line 485: 30 degree should not be called room temperature.
Author Response
- Line 94: the authors state that starvation and Rapa treatment does not affect cell viability, which is in contrast to the data shown in Fig. 1D and 2A.
Answer: First of all, thank you very much for your suggestions. Secondly, I apologize for the misunderstanding caused by the lack of rigor in our description. After serum starvation and rapamycin treatment of adipocytes, we examined cell viability. Although the cell viability had decreased after serum starvation for 8h and 100nM Rapa treatment for 12h, it did not reach significant levels (p＞0.05). Therefore, we chose 8 hours of serum starvation and 12 hours of rapamycin treatment as the time nodes for the next experiment.
Line 87-88, we exchanged “The results showed that cell viability was not changed by serum starvation and Rapa treatment (Figure 1D, 2A)” to “The results showed that serum starvation for 8h and 100nM Rapa treatment for 12h did not change cell viability (Figure 1D, 2A) (p> 0.05)”.
- To evaluate GFP-positive puncta, the transfection efficiency and stability is of major importance. Has that been checked?
Answer: Thank you very much for your suggestions. Before conducting formal experiments, we tested the transfection efficiency and stability of GFP. We tested both the first-transfected cells and the cells after passage. The results showed as follows: The transfection efficiency of GFP in the first generation cells was above 95%. Moreover, the transfection efficiency after passaging 3 and 5 generations is still above 90%, which proves that it can be stably inherited. Since this result is not the main content of the experiment, we did not put it in the manuscript.
- Browning effects should also be studied by induced browning of 3T3-L1 cells upon beta-receptor stimulation, which is more relevant to the in vivo situation.
Answer: Thank you very much for your suggestions. These suggestions are very important for us to improve the content of the manuscript. According to the reviewer's suggestion, we used β3-adrenergic receptor (β3-AR) agonists to pretreat adipocytes and tested the effect of treatment (not shown in the manuscript) (1) and re-examine the expression of browning-related genes in white adipose tissue. The specific results were shown
in Figure 7 C-F (C-F).
Line393-394, we added “in the case of β3-adrenergic receptor agonist (β3-AR) pretreatment”
- Line 485: 30 degree should not be called room temperature.
Answer：Thank you for your careful observation. We are sorry for our mistake. We have made changes in the manuscript. Thanks again.
Line 546, we exchanged “room temperature” to “normal temperature”
Other amendments:
Line 9, we exchanged “increased” to “increases”.
Line 13, we exchanged “. Under these two model conditions, we” to “and”.
Line 14, we added “significantly”.
Line 15, we deleted “significantly” and added “tests”.
Line 17, we deleted “pathway” and added “Moreover,”.
Line 27, we deleted “in”.
Line 32, we exchanged “are” to “were”.
Line 33, we added “research” and “that”.
Line 34, we exchanged “promotes” to “promoted”.
Line 35, we exchanged “triggers” to “triggered”.
Line 36, we exchanged “promotes” to “promoted”.
Line 38, we exchanged “established” to “establish” and added “a”.
Line 39, we exchanged “roles” to “role”.
Line 40, we deleted “or self-eating”.
Line 56, we deleted “Macroautophagy has been widely studied and profound form of autophagy [14,15,16,17,18]” and added “-induced loss of lipid droplets was”.
Line 57, we added “, whereas the” and “increased”
Line 58, we exchanged “19” to “14” and exchanged “inhibited” to “inhibits”.
Line 59, we exchanged “decreased” to “decreases” and exchanged “An” to “The”.
Line 60, we deleted “mouse” and exchanged “generated” to “generates” and exchanged “lean mice with decreased white adipose mass and enhanced” to “enhancement of” and added “the”.
Line 61, we added “mouse” and “, resulting in the lower of white adipose mass”.
Line 62, we exchanged “20” to “15” and added “the” and added “and essential for the” and deleted “during an”.
Line 63, we exchanged “21” to “16”.
Line 64, we exchanged “induces” to “ induced”.
Line 65, we exchanged “22” to “17”.
Line 66, we exchanged “inhibits” to “inhibited”.
Line67, we exchanged “23” to “18”.
Line68, we exchanged “had” to “have a” and exchanged “were” to “been”.
Line 69, we exchanged “was” to “is”
Line 71, we exchanged “24” to “19” and deleted “can” and exchanged “inhibit” to “inhibited”.
Line 73, we exchanged “25” to “20”.
Line 74, we exchanged “between” to “among” and deleted “and”.
Line 75, we exchanged “26,27” to “21,22”.
Line 76, we exchanged “And” to “Furthermore,”.
Line 77, we added “that”.
Line 79, we deleted “providing a theoretical basis for fat metabolism-related diseases,” and exchanged “the” to “a” and added “strategy” and deleted “of Mark4 as a drug” and added “and”.
Line 86, we deleted “of the cells are” and added “were”.
Line 89, we deleted “that of”.
Line 90, we exchanged “is” to “was”.
Line 91, we added “was” and exchanged “associates” to “associated”.
Line 261, we exchanged “concluded” to “conclude”.
Line 283, we exchanged “changes in” to “occurrence of”.
Line 292, we exchanged “but” to “whereas”.
Line 293, we exchanged “higher” to “elevated”.
Line 294, we deleted “that of” and deleted “under serum starvation”.
Line 296, we deleted “expression and”.
Line 297, we deleted “can” and exchanged “affect” to “affects”.
Line 306, we exchanged “was” to “were”.
Line 307, we exchanged “were” to “was”.
Line 309, we exchanged “treatment” to “treating”.
Line 343, we exchanged “confirms” to “confirmed” and exchanged “promotes” to “promoted”.
Line 345, we added “The” and added “that”.
Line 346, we exchanged “that of” to “the”.
Line 348, we deleted “higher”.
Line 350, we deleted “that of”.
Line 352, we exchanged “After” to “By”.
Line 354, we exchanged “after” to “through”.
Line 355, we exchanged “mTOR” to “mTOR-”.
Line 358, we added “, with”.
Line 380, we exchanged “between” to “among”.
Line 382-383, we deleted “on the basis of serum starvation treatment” and added “on the basis of serum starvation treatment with smaller” and deleted “but the” and deleted “became smaller”.
Line 395, we exchanged “After” to “By”.
Line 399, we exchanged “expression” to “expressions”.
Line 401, we exchanged “this” to “these” and exchanged “conclusion” to “findings”.
Line 402, we exchanged “are” to “were”.
Line 403, we added “were”.
Line 429, we added “were”.
Line 432, we exchanged “conclude” to “concluded”.
Line 433, we exchanged “on” to “in”.
Line 447, exchanged “adipocyte” to “adipocytes”.
Line 448, we added “it is”.
Line 452, we added “, which”.
Line 454, we exchanged “28” to “23”.
Line 458, we exchanged “promotes” to “promoted”.
Line 459, we exchanged “And the” to “The”.
Line 460, we exchanged “were” to “are”.
Line 461, we exchanged “established” to “establish”.
Line 462, we exchanged “on” to “in”.
Line 464, we exchanged “16” to “24” and added “the”.
Line 467, we exchanged “17” to “25” and exchanged “speculated” to “speculate”.
Line 471, we deleted “successfully”.
Line 479, we exchanged “29,30” to “26,27”.
Line 481, we exchanged “30,31” to “28,29”.
Line 482, we exchanged “treated” to “treating”.
Line 486, we added “that”.
Line 491, we exchanged “33” to “30”.
Line 492, we added “the”.
Line 493, we exchanged “34,35” to “31,32”.
Line 511, we exchanged “amount” to “number” and exchanged “increased” to “increases” and exchanged “36,37” to “33,34”.
Line 512, we exchanged “seemed” to “seem”.
Line 513, we exchanged “38” to “35”.
Line 514, we exchanged “39” to “36”.
Line 517, we exchanged “is” to “was”.
Line 519, we exchanged “40,41,42” to “37,38,39” and exchanged “And the” to “This”.
Line 569, we exchanged “43” to “40”.
Line 582, we exchanged “25” to “20”.
Round 2
Reviewer 2 Report
In general, my concerns have been relieved. However, transfection efficiency should be stated within the methods section. Further, results from b3-stimulation needs to be described and discussed in more detail.
Author Response
Answer to reviewers:
- However, transfection efficiency should be stated within the methods section.
Answer: Thank you very much for your suggestions. We have explained the transfection efficiency and stability in detail in the "Materials and methods" section of the manuscript.
Line 389-391, we added “The cells transfected with the GFP-LC3 plasmid were continuously passed to 5 generations, and the fluorescence expression of the 1st, 3rd and 5th generation cells was detected to determine the efficiency and stability of transfection (Results are not shown)”.
- Further, results from b3-stimulation needs to be described and discussed in more detail.
Answer: Thank you for your suggestions, which greatly help us improve the content of the manuscript.
Line 190-198, we added “In order to further explore the effect of Mark4 on the browning of white adipose tissue during starvation-induced autophagy, we first constructed the browning model by treating adipocytes with β3-adrenergic receptor agonist (β3-AR) while β3-AR agonist is considered to be a classic reagent for building browning models [23]. The results showed that mRNA and protein expression of the marker genes of brown adipose tissue increased significantly after treating with β3-AR treatment (P <0.01), proving that the browning model was successfully constructed (Figure 7C, D). Next, we examined the effect of Mark4 on the browning of white adipose tissue during autophagy in the case of β3-AR pretreatment.” And we deleted “In order to further explore the effect of Mark4 on the browning of white adipose tissue during starvation-induced autophagy, we examined the genes regulated to browning of white adipose tissue in the case of β3-adrenergic receptor agonist (β3-AR) pretreatment”.
Line 313-315，we added “It is a classic method to study browning by building a model, while β3-AR agonist has been considered as a classic reagent for building a browning model [38].”
- Richard, J. E.; López-Ferreras, L.; Chanclón, B.; Eerola. K.; Micallef, P.; Skibicka, K. P.; Wernstedt Asterholm, I. CNS β-adrenergic receptor activation regulates feeding behavior, white fat browning, and body weight. Am. J. Physiol. Endocrinol. Metab 2017, 313: E344-E358.
- Gavaldà-Navarro, A.; Moreno-Navarrete, J. M.; Quesada-López, T.; Cairó, M,; Giralt, M.; Fernández-Real, JM.; Villarroya, F. Lipopolysaccharide-binding protein is a negative regulator of adipose tissue browning in mice and humans. Diabetologia 2016, 59: 2208-18.
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
Yang et al and colleagues proposed that Mark4 is the negative regulator of browning of white adipose tissue. Mark4 knockout mice resist against obesity phenotype. It would be much more evitable if they examined the readily available KO mice in their study. Most of their mechanism study was done in 3T3-L1 preadipocytes which is not even adipose cells yet but fibroblasts without fat. In this regards, 1) they need to study browning markers after differentiating 3T3-L1 in Mark4 knockdown or overexpression and 2) examine the browning markers in adipose tissues (EWAT, SubQ and BAT) in Mark4 KO mice instead of using primary adipose cells from EWAT of wild type mice. Even it is desirable to isolate adipose primary cells from subcutaneous white adipose tissue which is more abundant in beige fat cells which is capable of either transdifferentiating into WAT or BAT lineage. In these major claws in the experimental design, this research paper dampens the interest of the reviewers, so it needs major revision in title and data for resubmission into other journals.
Author Response
- Most of their mechanism study was done in 3T3-L1 preadipocytes which is not even adipose cells yet but fibroblasts without fat. And they need to study browning markers after differentiating 3T3-L1 in Mark4 knockdown or overexpression.
Answer：First of all, thank you for your suggestions. Secondly, I apologize for the misunderstanding that has been caused to you by our negligence. The cells we used in this experiment were 3T3-L1 pre-adipocytes. After they were cultured for a certain period of time, they were induced to differentiate into mature adipocytes. Then relevant experimental research is being carried out. This is a very mature technology for us. Specific methods of inducing differentiation have been updated in the "Materials and Methods" section of the manuscript [1].
Line 501-527, we added the “Mouse 3T3-L1 pre-adipocytes were inoculated at a concentration of 1 × 106/ml and cultured in Dulbecco’s modified Eagle’s medium (DMEM) high glucose (Gibco) with 10% fetal bovine serum (Sigma) at 37°C and 5% CO2. Confluent cells were differentiated at day 0 in DMEM high glucose with 10% FBS, 10µg/ml bovine insulin (Sigma), 1 µM dexamethasone (Sigma), 0.5 mM isobutyl-1-methylxanthine (Sigma). On day 2, media was changed with DMEM high glucose, 10% FBS and 10 µg/ml bovine insulin. Day 4 and afterwards, cells were cultured in DMEM high glucose plus 10% FBS.”
[1] Feng, M.; Tian, L.; Gan, L.; Liu, Z.J.; Sun, C. Mark4 promotes adipogenesis and triggers apoptosis in 3T3-L1 adipocytes by activating JNK1 and inhibiting p38MAPK pathways. Biology of the Cell 2014, 106, 294-307.
- Examine the browning markers in adipose tissues (EWAT, SubQ and BAT) in Mark4 KO mice instead of using primary adipose cells from EWAT of wild type mice.
Answer：Thank you for your suggestions. In this experiment, the purpose of the in vivo experiment was to examine the effects of cold stimulation and HFD feeding on Mark4 expression, autophagy and browning of white adipose tissue in mice. Therefore, we used wild mice for experiments and did not modify them in any way. This is a very appropriate suggestion for the Mark4 KO mice you mentioned. We will adopt your suggestions in subsequent experiments to make the results more accurate.
- Even it is desirable to isolate adipose primary cells from subcutaneous white adipose tissue which is more abundant in beige fat cells which is capable of either transdifferentiating into WAT or BAT lineage.
Answer：Thank you for your suggestion. Before conducting the experiments described in the manuscript, we consulted a large amount of literature. From these literatures, we found that primary adipose cells can be isolated from subcutaneous white adipose tissue [1]. Therefore, we also refer to these results to isolate primary cells from subcutaneous white adipose tissue for related experiments.
[1] Auger, C.; Knuth, C.M.; Abdullahi, A.; Samadi, O.; Parousis, A.; Jeschke, M.G. Metformin prevents the pathological browning of subcutaneous white adipose tissue. Mol Metab 2019, 29, 12-23.
Reviewer 2 Report
In the manuscript titled “Mark4 inhibited the browning of white adipose tissue by promoting adipocytes autophagy in mice”, Yang et. reported that the MAP/microtubule affinity regulating kinase 4 (MARK4) regulates autophagy in adipocytes. The authors provide evidences that the regulatory effects of MARK4 on autophagy occurs through an increase of the activity of AMPK and a decrease of the AKT/mTOR signaling during serum starvation. It has been previously described that a decrease of the autophagic activity in adipocytes is associated with an increase in the thermogenic activity of brown adipose tissue (BAT) and an increase in the browning of white adipose tissue (WAT). The authors shown that MARK4 expression was downregulated upon cold exposure in BAT and WAT, and upregulated after a high fat diet. The authors propose a model in which MARK4 activity negatively modulates browning by inducing autophagy. The results are potentially interesting however the manuscript in its current form lacks direct evidences of MARK4 activity in the inhibition of browning. Some data and experimental procedures presented in this manuscript should be clarified, whereas additional experiments should be carried out.
Concerns with the manuscript:
The “Introduction” section of the manuscript requires extensive revision. The existing literature should be better presented (in a way that justifies the aim of the study) and it should be more coherent with the essence of the study. I would also suggest a revision and reorganization of the “introduction” and “discussion” sections.
The methodology should be better justified.
In the first paragraph the cell viability tests performed should be better explained. If the assays were performed in order to choose the timings and concentrations in which there were no statistical differences between treatments, this should be better explained in the text. After 10 h of serum starvation there was a significant decrease in cell viability, as well as after 16h of 100nM rapamycin treatment.
The authors should clarify when the cells are differentiated adipocytes or pre-adipocytes, as well as the cell line used in all the experiments (consider also include this information in all figure legends). In the first paragraph the authors indicated that 3T3-L1 pre-adipocytes were used whereas in the materials and methods section there’s a section describing white adipose tissue primary cultures (non-differentiated, cultured as pre-adipocytes). In this sense, its important to clarify if the results were obtained using precursor cells or using differentiated adipocytes and thus correct if necessary, some statements and conclusions. This can also be applied to the extrapolated conclusions from the in vitro experiments: my major concern is that there is no evidence in the data presented in this manuscript supporting that the process of browning is affected by the changes in Mark4 expression; browning needs to be tested in vivo (see below), otherwise the assertion that Mark4 expression is affecting browning should be toned down using a more speculative sentence.
Are the levels of Mark4 affected by the starvation and rapamycin treatments?
LC3A and LC3B are two different proteins. The correct nomenclature for the cytosolic LC3B is LC3B-I whereas the autophagosome-associated LC3B (conjugated with phosphatidylethanolamine) is LC3B-II. This mistake should be amended in the manuscript, in the figures and in the abstract. Moreover, please clarify in which western blot the quantification of LC3B-II is expressed as the ratio LC3B I/II. The ratio is mentioned in the manuscript whereas LC3B-II protein levels quantification appears to have been normalized using the loading control in the figures. The correct way to present LC3B-II quantification is using the loading control for normalization.
Figures 1D, 2D, 3B, 5B: MCD and DAPI staining show very strong colocalization patterns, thus the MCD signal seems to be an artefact. The authors should clarify this or repeat the assays. Please check the labels in Figure1D and Figure 2D.
In the text the authors refer to endogenous LC3 detection whereas in the figures the panels showing LC3 fluorescence are labeled as GFP-LC3. Have the assays being performed using a GFP-LC3 expression vector? In this case the signal would be not reflecting the endogenous LC3. In any case the presence of cytoplasmic LC3 does not reflect the amount of autophagosomes. LC3 immunostaining should be interpreted as a surrogate marker of the presence of autophagosome only when distributed as puncta, and data should be expressed as LC3 puncta per cell.
The images corresponding to Mark4 overexpression seem to contain more cells (in both GFP-LC3 and MCD). The authors should show DAPI staining also for the GFP-LC3 images. Please clarify this or if necessary, repeat the experiments for a correct presentation and interpretation of the data.
Figure 4: check the legend in the histogram of the panel B. The quantification of p62 in cells treated with compound C do not reflect the bands appearing in the blot.
Has p-AKT been analyzed using adipocytes pre-treated with insulin? Add this to the figure or remove it from the legend. In any case, the use of insulin should be justified in the text.
In the in vivo experiments, have the control mice been exposed to 37ºC for 24h? The thermoneutral temperature for mice is around 30ºC. When using thermoneutrality as the control temperature, mice should have been previously acclimated, otherwise both groups (thermoneutrality and cold exposed mice) are experiencing changes in BAT and WAT due to the acclimation to the new environment. If this can not be amended, the use of mice kept at room temperature as controls (animal facility temperature) is more suitable than non-fully acclimated mice.
Serum starvation and rapamycin treatment may not be the best model to analyze the process of browning. The induction of browning (in vitro) can be promoted using ppargamma agonists or activators of the beta3-adrenergic pathway.
Taking in account the current data, the interaction between autophagy activators (starvation and rapamycin), autophagy inhibitors (3-MA), and the modulators of the expression of Mark4 (vectors), should be better explained and discussed.
In figure 8 the effects of rapamycin/starvation on the expression levels of the thermogenic genes and proteins should be added for a better interpretation of the results. The authors claim that the treatments alone (serum starvation and rapamycin) did not affect the expression on thermogenic genes without the appropriate control of the experiment. And, as said above, the extrapolation of these results obtained in vitro to the physiology of the adipose tissue should be toned down unless additional in vivo experiments are performed. If it is possible, consider the use of Mark4-KO mice.
There exists literature (even revisions) regarding autophagy and browning/thermogenesis/mTOR/rapamycin, the authors should add some of those references in the discussion.
Materials and methods:
Provide more accurate information about the generation of the vector pcDNA3.1-Mark4. If it has been previously published, please add the corresponding reference. Provide information about the GFP-LC3 plasmid. Cell culture: 3t3-L1 cell culture methods is missing. Please clarify if the cells have been differentiated or maintained as precursors.
General comments:
The English grammar and syntaxis need to be corrected.
Correct the mistakes in Figure legends and labels.
Check abbreviations and nomenclature.
Minor comments
In the introduction Mark4 is mentioned also as Par-1, this can be confusing. The explanation of the results regarding Ref 25 and 28 are mixed in line 63 and 64 (Ap2-driven and Myf5-driven genetic invalidation of Atg7). Correct the concept “muscle beige cells”.
Are Figure 5A and Figure 3A the same?
Figure 9: check the arrows, mTOR is an autophagy inhibitor. Why is Mark4 outside the cell? The design of the scheme needs to be improved: the treatments (rapamycin and starvation) and the intracellular effectors are unconnected.
Author Response
- The “Introduction” section of the manuscript requires extensive revision. The existing literature should be better presented (in a way that justifies the aim of the study) and it should be more coherent with the essence of the study. I would also suggest a revision and reorganization of the “introduction” and “discussion” sections.
Answer：We are very thankful for your time and efforts. Your advice is very helpful and meaningful to make this manuscript better. At your suggestion, we revised and reorganized the “Introduction” and “Discussion” of the manuscript. And these changes have been highlighted by the Track Changes functionality within Word in the revised manuscript.
Line 25, we exchanged the “The main difference is” to “which difference in”.
Line 27, we exchanged the “differentiation” to “in the differentiation of”.
Line 28-30, we deleted “As the AMPK-related family of kinases, physiological functions of the Mark2 and Mark3 kinase have recently been studied by using targeted gene knockout approaches in mice. The Mark2 pathway involves a variety of physiological processes including fertility, immune system homeostasis, glucose homeostasis, energy metabolism, learning, memory, etc. [7-9] In addition to Mark2, most information regarding the cell biological functions of the Par-1 kinase comes from studies of Mark3. Studies have shown that MARK3 may be closely related to osteoporosis, obesity, and the construction of cell polarity [10,11]” and added the “Studies indicate that Mark4 is the negative regulator of mTORC1 which plays a central role in cell growth [7,8]”.
Line 31-32, we added “protein kinase B (PKB/ AKT)”and “AMP-activated protein kinase (AMPK)”
Line 33, we exchanged “12” to “9”.
Line 34, we added “Jun N-terminal kinase (JNK)” and “mitogen-activated protein kinase (MAPK)”
Line 36, we exchanged “13” to “10” and added the “that”.
Line 37-38, we added the “peroxisome proliferator-activated receptor gamma (PPARγ)” and the “nuclear factor-kappa B(NF-κB)”.
Line 39, we exchanged “14” to “11”
Line 61, we added the “has been” and deleted “is the most” and exchanged “15,16” to “12,13”.
Line 62, we exchanged “17,18,19,20,21” to “14,15,16,17,18”.
Line 63, we added the “the”.
Line 64, we exchanged “22” to “19” and added the “autophagy-related gene 7 (Atg7)”
Line 68, we exchanged “23” to “20”
Line 69, we exchanged “24” to “21”
Line 71, we exchanged “impairment of” to “impairing” and exchanged “prevents” to “preventing” and added the “HFD”
Line 72, we exchanged “inhibited” to “inhibites” and exchanged “prevents” to “preventing”
Line 73, we exchanged “regulate on” to “regulating expression” and exchanged “26” to “23” an
Line 77, we exchanged “27” to “24”
Line 78, we added “Activating transcription factor 4(Atf4)” and “autophagy-related gene 5(Atf5)”
Line 79, we exchanged “29” to “25”
Line 80-81, we added “At present, the relationship between Mark4 and autophagy and browning of white adipose tissue has not been reported in adipose tissue [26,27].”
Line 79, we added “which”
Line 390, we deleted “s”
Line 392, we exchanged “30” to “28”
Line 402, we exchanged “19” to “16”
Line 403, we deleted “of”
Line 404, we exchanged “20” to “17”
Line 411, we exchanged “LC3B” to “LC3B-II”
Line 416, we exchanged “31,32” to “29,30”
Line 418, we exchanged “33,34” to “31,32”
Line 424, we deleted “independent on AMPK/AKT signaling pathways.”
Line 428, we exchanged “35” to “33”
Line 429, we deleted “, another relevant component of the “adipose organ” ,” and added “of” and added “ing”
Line 430, we exchanged “36,37” to “34,35”
Line 431, we exchanged “38,39” to “36,37”
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Line 477, we exchanged “41” to “39” and exchanged “Therefore, we studied the role of Mark4 in regulating the browning of white adipose tissue through inhibiting autophagy. O” to “In our studies, the o”
Line 482, we exchanged “42,43,44” to “40,41,42”
- In the first paragraph the cell viability tests performed should be better explained. If the assays were performed in order to choose the timings and concentrations in which there were no statistical differences between treatments, this should be better explained in the text. After 10 h of serum starvation there was a significant decrease in cell viability, as well as after 16h of 100nM rapamycin treatment.
Answer: Thank you for your suggestion. Before formal experiments, we performed CCK8 assays on cell viability after serum starvation and rapamycin treatment, and oil red O staining of adipocytes (below). Although the cell viability decreased slightly after 8 hours of serum starvation, it did not reach the significant standard. Similarly, the decrease in cell viability after 100 nM rapamycin treatment for 8 hours did not reach a significant standard. Therefore, in the subsequent experiments, we chose to perform relevant detection after 8 hours of processing (Figure 1A, 2A).
- The authors should clarify when the cells are differentiated adipocytes or pre-adipocytes, as well as the cell line used in all the experiments (consider also include this information in all figure legends). In the first paragraph the authors indicated that 3T3-L1 pre-adipocytes were used whereas in the materials and methods section there’s a section describing white adipose tissue primary cultures (non-differentiated, cultured as pre-adipocytes). In this sense, its important to clarify if the results were obtained using precursor cells or using differentiated adipocytes and thus correct if necessary, some statements and conclusions.
Answer：First of all, thank you for your suggestions. Secondly, I apologize for the misunderstanding that has been caused to you by our negligence. The cells we used in this experiment were 3T3-L1 pre-adipocytes. After they were cultured for a certain period of time, they were induced to differentiate into mature adipocytes. Then relevant experimental research is being carried out. This is a very mature technology for us. Specific methods of inducing differentiation have been updated in the "Materials and Methods" section of the manuscript [1].
Line 501-527, we added the “Mouse 3T3-L1 pre-adipocytes were inoculated at a concentration of 1 × 106/ml and cultured in Dulbecco’s modified Eagle’s medium (DMEM) high glucose (Gibco) with 10% fetal bovine serum (Sigma) at 37°C and 5% CO2. Confluent cells were differentiated at day 0 in DMEM high glucose with 10% FBS, 10µg/ml bovine insulin (Sigma), 1 µM dexamethasone (Sigma), 0.5 mM isobutyl-1-methylxanthine (Sigma). On day 2, media was changed with DMEM high glucose, 10% FBS and 10 µg/ml bovine insulin. Day 4 and afterwards, cells were cultured in DMEM high glucose plus 10% FBS.”
[1] Feng, M.; Tian, L.; Gan, L.; Liu, Z.J.; Sun, C. Mark4 promotes adipogenesis and triggers apoptosis in 3T3-L1 adipocytes by activating JNK1 and inhibiting p38MAPK pathways. Biology of the Cell 2014, 106, 294-307.
- This can also be applied to the extrapolated conclusions from the in vitro experiments: my major concern is that there is no evidence in the data presented in this manuscript supporting that the process of browning is affected by the changes in Mark4 expression; browning needs to be tested in vivo (see below), otherwise the assertion that Mark4 expression is affecting browning should be toned down using a more speculative sentence
Answer: Thank you for your suggestion. Many previous studies have shown that UCP1, PGC1α, and PRDM16 are important factors related to browning of white adipose tissue. Changes in the expression of these factors are largely representative of browning [1-3]. In this manuscript, we examined the effect of changes in Mark4 expression on browning-related factors in white adipose tissue from the mRNA and protein levels. To some extent, Mark4 has been proved to be related to browning.
[1] Dempersmier J , Sambeat A , Gulyaeva O , et al. Cold-Inducible Zfp516 Activates UCP1 Transcription to Promote Browning of White Fat and Development of Brown Fat[J]. Molecular Cell, 2015, 57(2):235-246.
[2] Huang L , Pan D , Chen Q , et al. Transcription factor Hlx controls a systematic switch from white to brown fat through Prdm16-mediated co-activation. Nature Communications 2017, 8, 68.
[3] Linyun, He, Mowei, et al. Obesity Associated miR-199a/214 Cluster Inhibits Adipose Browning via PRDM16-PGC-1α Transcriptional Network. Diabetes 2018.
- Are the levels of Mark4 affected by the starvation and treatments?
Answer: Thank you very much for your suggestions. We reviewed the article and found that there
are no relevant studies on the effects of serum starvation and rapamycin treatment on Mark4 expression. Therefore, we carried out relevant tests through experiments. The results showed that
neither starvation nor rapamycin treatment affected Mark4 expression.
- LC3A and LC3B are two different proteins. The correct nomenclature for the cytosolic LC3B is LC3B-I whereas the autophagosome-associated LC3B (conjugated with phosphatidylethanolamine) is LC3B-II. This mistake should be amended in the manuscript, in the figures and in the abstract.
Answer：Thank you for your suggestion. We have made changes in manuscript, figures and abstract.
Line 16、96、97、194、205、207、210、212、236、238、255、411、Figure1C、Figure2C、Figure3E、Figure4B、4D、Figure5D，we exchanged “LC3B” to “LC3B-II”
- Moreover, please clarify in which western blot the quantification of LC3B-II is expressed as the ratio LC3B I/II. The ratio is mentioned in the manuscript whereas LC3B-II protein levels quantification appears to have been normalized using the loading control in the figures. The correct way to present LC3B-II quantification is using the loading control for normalization.
Answer: Thank you for your suggestion. The quantification of LC3B-II in Figures 1C and 2C is expressed as the ratio of LC3B I / II. The specific results are shown below. (Left: serum starvation; right: rapamycin).
- Figures 1D, 2D, 3B, 5B: MCD and DAPI staining show very strong colocalization patterns, thus the MCD signal seems to be an artefact. The authors should clarify this or repeat the assays.
Answer: Thank you for your suggestion. Monodansylcadaverine (MDC) has been widely used as a specific marker for autophagic vacuoles, since it was shown to accumulate in acidic compartments enriched in lipids [1]. MDC staining can visualize the location of autophagic vesicles. DAPI staining shows the location of the nucleus. Previous studies have shown that autophagosomes displayed by MDC staining appear around the nucleus. Our experimental results show the same. We repeated this experiment, and the results are consistent with the results described in the manuscript.
[1] Bampton E T W , Goemans C G , Niranjan D , et al. The Dynamics of Autophagy Visualised in Live Cells: from Autophagosome Formation to Fusion with Endo/lysosomes[J]. Autophagy, 2005, 1(1):23-36.
- Please check the labels in Figure1D and Figure 2D.
Answer: Thank you for your suggestion. We have modified the labels in Figures 1D and 2D. The original picture has also been replaced in manuscript.
- In the text the authors refer to endogenous LC3 detection whereas in the figures the panels showing LC3 fluorescence are labeled as GFP-LC3. Have the assays being performed using a GFP-LC3 expression vector? In this case the signal would be not reflecting the endogenous LC3. In any case the presence of cytoplasmic LC3 does not reflect the amount of autophagosomes. LC3 immunostaining should be interpreted as a surrogate marker of the presence of autophagosome only when distributed as puncta, and data should be expressed as LC3 puncta per cell.
Answer: Thank you very much for your attention to this detailed issue. In this experiment, the purpose of using the GFP-LC3 expression vector is to prove the occurrence of autophagy, rather than to detect the endogenous LC3 production. The results are shown in Figure 3B and Figure 5B. We reselected a different field of view instead of the previous picture to represent the generation of autophagosomes.
- The images corresponding to Mark4 overexpression seem to contain more cells (in both GFP-LC3 and MCD).
Answer: Thank you for your suggestion. We are very sorry for the misunderstanding caused by our choice of vision. We reselected a different field of view and replaced the original picture. Thanks again for your suggestions (Figure3B and Figure5B)
- The authors should show DAPI staining also for the GFP-LC3 images. Please clarify this or if necessary, repeat the experiments for a correct presentation and interpretation of the data.
Answer: Thank you for your suggestion. The results presented in this manuscript are the results of repeated experiments, and we have selected the best quality pictures to present here. Due to the short modification time, we will supplement the DAPI-stained pictures in subsequent experiments.
- Figure 4: check the legend in the histogram of the panel B. The quantification of p62 in cells treated with compound C do not reflect the bands appearing in the blot.
Answer: Thank you for your suggestion. Our experimental results in Figure 4B were re-tested by Western blot, and the original experimental results were replaced. Thanks again for your suggestions.
- Has p-AKT been analyzed using adipocytes pre-treated with insulin? Add this to the figure or remove it from the legend. In any case, the use of insulin should be justified in the text.
Answer: Thank you for your question. We apologize for the unnecessary trouble we have caused to you. We did not use insulin in this experiment, and we have also modified the relevant content.
Line 236, we deleted the “or insulin”.
15.In the in vivo experiments, have the control mice been exposed to 37ºC for 24h? The thermoneutral temperature for mice is around 30ºC. When using thermoneutrality as the control temperature, mice should have been previously acclimated, otherwise both groups (thermoneutrality and cold exposed mice) are experiencing changes in BAT and WAT due to the acclimation to the new environment. If this can not be amended, the use of mice kept at room temperature as controls (animal facility temperature) is more suitable than non-fully acclimated mice.
Answer: First of all, thank you very much for your attention to this detailed issue. Secondly, I apologize for our clerical errors. Before cold exposure, both groups of mice were acclimatized for 24 hours at 30 ° C to ensure that they adapted to the new environment. After that, one group of mice continued to survive at 30 ° C for 24 hours, and another group of mice survived at 4 ° C for 24 hours [1].
Line 497, we exchanged “37” to “30”
[1] Xiao L , Ru J , Qiangling Z , et al. Cold-Induced Browning Dynamically Alters the Expression Profiles of Inflammatory Adipokines with Tissue Specificity in Mice[J]. International Journal of Molecular Sciences, 2016, 17(5):795-.
- Serum starvation and rapamycin treatment may not be the best model to analyze the process of browning. The induction of browning (in vitro) can be promoted using ppargamma agonists or activators of the beta3-adrenergic pathway.
Answer: Thank you for your suggestion. In this experiment, we used serum starvation and rapamycin treatment to construct an autophagy model. Serum starvation and rapamycin treatment have proven to be classic methods for building autophagy models [1-2].
[1] Barutcu S A , Girnius N , Vernia S . Role of the MAPK/cJun NH-Terminal Kinase signaling pathway in starvation-induced autophagy.[J]. International Journal of Molecular Medicine, 2018, 42(1).
[2] Vizza D , Perri A , Toteda G , et al. Rapamycin-induced autophagy protects proximal tubular renal cells against proteinuric damage through the transcriptional activation of the nerve growth factor receptor NGFR[J]. Autophagy.
- Taking in account the current data, the interaction between autophagy activators (starvation and rapamycin), autophagy inhibitors (3-MA), and the modulators of the expression of Mark4 (vectors), should be better explained and discussed.
Answer: Thank you for your suggestion. In this experiment, we constructed the autophagy model of adipocytes by serum starvation and rapamycin treatment [1-2]. Based on the autophagy model, we constructed Mark4 overexpression and interference vectors to regulate the expression of Mark4, and examined the expression of white adipose tissue browning-related genes to investigate the effect of Mark4 on white adipose tissue browning. The use of autophagy inhibitors (3-MA), and the modulators of the expression of Mark4 (vectors) allows us to further determine the relationship between Mark4 and autophagy [3]. Therefore, by using autophagy activators, autophagy inhibitors (3-MA), and the modulators of the expression of Mark4 (vectors), we accurately determined the relationship between Mark4, autophagy, and browning of white adipose tissue.
[1] Barutcu S A , Girnius N , Vernia S . Role of the MAPK/cJun NH-Terminal Kinase signaling pathway in starvation-induced autophagy.[J]. International Journal of Molecular Medicine, 2018, 42(1).
[2] Vizza D , Perri A , Toteda G , et al. Rapamycin-induced autophagy protects proximal tubular renal cells against proteinuric damage through the transcriptional activation of the nerve growth factor receptor NGFR[J]. Autophagy.
[3] Wu Y , Wang X , Guo H , et al. Synthesis and screening of 3-MA derivatives for autophagy inhibitors[J]. Autophagy, 2013, 9(4):595-603.
- In figure 8 the effects of rapamycin/starvation on the expression levels of the thermogenic genes and proteins should be added for a better interpretation of the results.
Answer: First of all, thank you for your valuable suggestions. Secondly, I apologize for the misunderstandings that have been caused to you by our mistakes. Because of our mistake, the relative protein expression level was incorrectly marked as the relative mRNA expression level in Figure8 E、F, which made the reviewer to believe that we did not have a protein level test result. We have modified the wrong figure (Figure8 E、F). We apologize again for this.
- The authors claim that the treatments alone (serum starvation and rapamycin) did not affect the expression on thermogenic genes without the appropriate control of the experiment. And, as said above, the extrapolation of these results obtained in vitro to the physiology of the adipose tissue should be toned down unless additional in vivo experiments are performed. If it is possible, consider the use of Mark4-KO mice.
Answer: Thank you for your suggestion. Rapamycin has been demonstrated to affect lipid metabolism through stimulating lipolysis, inhibiting de novo lipogenesis and reducing adiposity [1]. However, there is currently no report on the effects of serum starvation on browning. The results of this experiment show that serum starvation does not affect the expression of thermogenic genes at the cellular level (Figure 8E), but rapamycin treatment affects the expression of thermogenic genes (Figure 8F). This is consistent with previously reported results. Regarding the Mark4-KO mice you mentioned, this has given us great inspiration, and we will adopt your suggestions in future experiments. Thanks again.
[1] Wang, Yan, Lie, et al. Chronic Rapamycin Treatment Improved Metabolic Phenotype but Inhibited Adipose Tissue Browning in High-Fat Diet-Fed C57BL/6J Mice[J]. Biological & pharmaceutical bulletin, 2017.
- There exists literature (even revisions) regarding autophagy and browning / thermogenesis / mTOR / rapamycin, the authors should add some of those references in the discussion.
Answer: Thank you very much for your suggestion, we have added literatures about autophagy and browning / thermogenesis / mTOR / rapamycin to the manuscript.
Line 28-30, we added “Studies indicate that Mark4 is the negative regulator of mTORC1 which plays a central role in cell growth [7,8].”
Line 80-81, we added “At present, the relationship between Mark4 and autophagy and browning of white adipose tissue has not been reported in adipose tissue [26,27].”
The numbers of the newly added references in the article are as follows:
[7] Li, L.; Guan, K. L. Microtubule-associated Protein/Microtubule Affinity-regulating Kinase 4 (MARK4) Is a Negative Regulator of the Mammalian Target of Rapamycin Complex 1 (mTORC1). Journal of Biological Chemistry 2013, 288, 703-708.
[8] Rovina, D.; Fontana, L.; Monti, L.; Novielli, C.; Panini, N.; Sirchia, S. M.; Erba, E. Magnani, I.; Larizza, L. Microtubule-associated protein/microtubule affinity-regulating kinase 4 (MARK4) plays a role in cell cycle progression and cytoskeletal dynamics. European Journal of Cell Biology 2014, 93, 355-365.
[26] Giampieri, F.; Afrin, S.; Forbes-Hernandez, T. Y.; Gasparrini, M.; Cianciosi, D.; Reboredo-Rodriguez, P.; Varela-Lopez, A.; Quiles, J. L.; Battino, M. Autophagy in Human Health and Disease: Novel Therapeutic Opportunities. Antioxidants & Redox Signaling 2018, ars,2017.7234-.
[27] Klionsky, D. J.; Abdelmohsen, K.; Abe, A.; Abedin, M. J.; Abeliovich, H.; Acevedo, Arozena. A.; Adachi, H.; Adams, C. M.; Adams, P. D.; Adeli, K. et al. Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy 2012, 8, 445-544.
- Provide more accurate information about the generation of the vector pcDNA3.1-Mark4. If it has been previously published, please add the corresponding reference.
Answer: Thank you for your suggestion. Accurate information about the generation of the vector pcDNA3.1-Mark4 has been added to the "Materials and Methods" section of the manuscript.
Line 554-568, we added “Transfection of adipocytes with plasmids. Mark4 forced expression plasmid vector HA-Mark4 was kept in our lab. shRNA sequence against Mark4 was contrived and synthesized by Genepharma Company (Shanghai, China) using pGPU6/Neo shRNA expression vector named sh1-Mark4, sh2-Mark4 and sh3-Mark4. Then by transfection efficiency detection, the optimal shRNA of Mark4 was chosen and named sh-Mark4. Plasmids vectors used as control vectors were pcDNA3.1-vector and negative-shRNA. To exclude off-target effects of shRNA treatment, we used the other two Mark4 shRNAs, sh1-Mark4 and sh3-Mark4 which targeting different sequences of Mark4 mRNA compared with shRNA-Mark45. Mark4 DA (dead mutant) was made as described previously7. In HA-Mark4 DA group, mark4 protein was translated inactively with specific amino acid mutations called dead mutant. 2 μg interference or expression plasmids DNA were mixed with 2 μl X-treme GENE HP Reagent (Roche, Switzerland) and Opti-MEMI media (Invitrogen, California, USA) and then added into the culture dish for 24 h or 48 h according to the protocol [11].”
[11] Liu, Z.; Gan, L.; Chen, Y.; Luo, D.; Zhang, Z.; Cao, W.; Zhou, Z.; Lin, X.; Sun, C. Mark4 promotes oxidative stress and inflammation via binding to PPARγ and activating NF-κB pathway in mice adipocytes. Scientific reports 2016, 6, 21382.
- Provide information about the GFP-LC3 plasmid.
Answer: Thank you for your suggestion. Information about GFP-LC3 has been added to the "Materials and Methods" section of the manuscript.
Line 547-554, we added “GFP-LC3 Analysis and Subcellular Localization. Cells were transfected with GFP-LC3 plasmid by using X-treme GENE HP Reagent (Roche, Switzerland) according to the manufacturer’s instructions. After 48 h transfection, cells were washed with OptiMEM I (Invitrogen, CA, USA, 51985042) and subjected to staining. The cells were stained with Lyso Tracker® Green DND probe (Thermo Scientific, CA, USA, L7526) as recommended by the manufacturer. The formation of GFP-LC3 punctate and Tracker fluorescence were visualized and analyzed using Cytation3 Cell Imaging Multi-Mode Reader (BioTek, VT, USA) [25].”
[25] Gan, L.; Liu, Z.; Luo ,D.; Ren, Q.; Wu, H.; Li, C.; Sun, C. Reduced endoplasmic reticulum stress-mediated autophagy is required for leptin alleviating inflammation in adipose tissue. Frontiers in immunology 2017, 8, 1507.
- Cell culture: 3t3-L1 cell culture methods is missing. Please clarify if the cells have been differentiated or maintained as precursors.
Answer: First of all, thank you for your suggestions. Secondly, I apologize for the misunderstanding that has been caused to you by our negligence. The cells we used in this experiment were 3T3-L1 pre-adipocytes. After they were cultured for a certain period of time, they were induced to differentiate into mature adipocytes. Then relevant experimental research is being carried out. This is a very mature technology for us. Specific methods of inducing differentiation have been updated in the "Materials and Methods" section of the manuscript [1].
Line 501-527, we added the “Mouse 3T3-L1 pre-adipocytes were inoculated at a concentration of 1 × 106/ml and cultured in Dulbecco’s modified Eagle’s medium (DMEM) high glucose (Gibco) with 10% fetal bovine serum (Sigma) at 37°C and 5% CO2. Confluent cells were differentiated at day 0 in DMEM high glucose with 10% FBS, 10µg/ml bovine insulin (Sigma), 1 µM dexamethasone (Sigma), 0.5 mM isobutyl-1-methylxanthine (Sigma). On day 2, media was changed with DMEM high glucose, 10% FBS and 10 µg/ml bovine insulin. Day 4 and afterwards, cells were cultured in DMEM high glucose plus 10% FBS.”
[1] Feng, M.; Tian, L.; Gan, L.; Liu, Z.J.; Sun, C. Mark4 promotes adipogenesis and triggers apoptosis in 3T3-L1 adipocytes by activating JNK1 and inhibiting p38MAPK pathways. Biology of the Cell 2014, 106, 294-307.
- The English grammar and syntaxis need to be corrected.
Answer: Answer: We are very thankful for your time and efforts. Your advice is very helpful and meaningful to make this manuscript better. We have checked the manuscript carefully to improve language description as well as language logic. And these changes and the other minor modifications of the manuscript has been highlighted by the Track Changes functionality within Word in the revised manuscript. Thanks again.
Line 94, we exchanged “was” to “were”
Line 99, we deleted “known to” and added “It is well known that”
Line 167, we added “cholecystokinin-8 (CCK8)”
Line 186, we exchanged “were” to “was”
Line 200, we exchanged “e” to “ing”
Line 256, we exchanged “were” to “was”
Line 261, we exchanged “was” to “were”
Line 285-385，we exchanged the order of results 7 and 8
Line 497, we exchanged “37” to “30”
The remaining changes are reflected in Questions 1 and 6.
- Correct the mistakes in Figure legends and labels.
Answer: Thank you for your suggestion. We have corrected the errors in Figure legends and labels.
The changes are reflected in Questions 1 and 6.
25 Check abbreviations and nomenclature.
Answer: Thank you for your suggestion. We have rewritten abbreviations and nomenclature in the manuscript.
Line 643-645, we exchanged the “Abbreviations” and “Nomenclature”
- In the introduction Mark4 is mentioned also as Par-1, this can be confusing. The explanation of the results regarding Ref 25 and 28 are mixed in line 63 and 64 (Ap2-driven and Myf5-driven genetic invalidation of Atg7). Correct the concept “muscle beige cells”.
Answer: Thank you for your suggestion. We have made changes in Introduction.
- Are Figure 5A and Figure 3A the same?
Answer: Thank you for your valuable suggestions. We have reanalyzed the Figure 5A and Figure 3A you mentioned. The results show that although the two figures are very similar, they are indeed obtained from different data analysis. Nevertheless, we also reanalyzed the results. The original results have also been replaced. We apologize for any misunderstandings. (Figure 5A and Figure 3A)
- Figure 9: check the arrows, mTOR is an autophagy inhibitor. Why is Mark4 outside the cell? The design of the scheme needs to be improved: the treatments (rapamycin and starvation) and the intracellular effectors are unconnected.
Answer: Thank you for your valuable suggestions. Your suggestions have played a key role in modifying and improving Figure 9. The modified Figure 9 has replaced the previous version. We have placed Mark4 inside cells and excluded serum starvation and rapamycin from the cells. Regarding the relationship between mTOR, Mark4 and autophagy, we have demonstrated that Mark4 can promote autophagy by inhibiting the mTOR pathway. Therefore figure 9 shows that Mark4 inhibits mTOR and promotes autophagy. Finally, thank you again for your valuable suggestions.
Reviewer 3 Report
The manuscript entitled "Mark4 inhibited the browning of white adipose tissue by promoting adipocytes autophagy in mice" reflects a considerable volume of work and analysis performed. It is well structured and quite well written. However, I have a major concern that should be considered before its publication in IJMS: authors emphasize on several occasions (throughout the document) that little is known about the role of MARK4 in autophagy, which is not true. The role of MARK4 in autophagy as well as its relation with the AMPK pathway are well known. In that sense, it is recommended to consult the following articles:
- DOI: 10.1080/15548627.2015.1100356;
- DOI: 10.1089/ars.2017.7234,
- DOI: 10.1074/jbc.C112.396903.
Therefore, in my opinion what could be highlighted in the paper is that autophagy models in adipocytes and the role of MARK4 in browning have been little studied.
Other minor concerns are:
- please write the abbreviations to the full extent the first time they appear in the text.
- please follow the same order in the material & methods and results sections (That is, in vitro analysis before and then in vivo analysis.
Author Response
1.The manuscript entitled "Mark4 inhibited the browning of white adipose tissue by promoting adipocytes autophagy in mice" reflects a considerable volume of work and analysis performed. It is well structured and quite well written. However, I have a major concern that should be considered before its publication in IJMS: authors emphasize on several occasions (throughout the document) that little is known about the role of MARK4 in autophagy, which is not true. The role of MARK4 in autophagy as well as its relation with the AMPK pathway are well known. In that sense, it is recommended to consult the following articles:
- DOI: 10.1080/15548627.2015.1100356;
- DOI: 10.1089/ars.2017.7234,
- DOI: 10.1074/jbc.C112.396903.
1.Therefore, in my opinion what could be highlighted in the paper is that autophagy models in adipocytes and the role of MARK4 in browning have been little studied.
Answer: We are very thankful for your time and efforts. Your advice is very helpful and meaningful to make this manuscript better. After reviewing the reference you provided, we are clearer about the relationship between Mark4, autophagy and browning. We have modified some sentences in the article to make it more accurate.
Line 28-30, we added “Studies indicate that Mark4 is the negative regulator of mTORC1 which plays a central role in cell growth [7,8].”
Line 80-81, we added “At present, the relationship between Mark4 and autophagy and browning of white adipose tissue has not been reported in adipose tissue [26,27].”
The numbers of the newly added references in the article are as follows:
[7] Li, L.; Guan, K. L. Microtubule-associated Protein/Microtubule Affinity-regulating Kinase 4 (MARK4) Is a Negative Regulator of the Mammalian Target of Rapamycin Complex 1 (mTORC1). Journal of Biological Chemistry 2013, 288, 703-708.
[8] Rovina, D.; Fontana, L.; Monti, L.; Novielli, C.; Panini, N.; Sirchia, S. M.; Erba, E. Magnani, I.; Larizza, L. Microtubule-associated protein/microtubule affinity-regulating kinase 4 (MARK4) plays a role in cell cycle progression and cytoskeletal dynamics. European Journal of Cell Biology 2014, 93, 355-365.
[26] Giampieri, F.; Afrin, S.; Forbes-Hernandez, T. Y.; Gasparrini, M.; Cianciosi, D.; Reboredo-Rodriguez, P.; Varela-Lopez, A.; Quiles, J. L.; Battino, M. Autophagy in Human Health and Disease: Novel Therapeutic Opportunities. Antioxidants & Redox Signaling 2018, ars,2017.7234-.
[27] Klionsky, D. J.; Abdelmohsen, K.; Abe, A.; Abedin, M. J.; Abeliovich, H.; Acevedo, Arozena. A.; Adachi, H.; Adams, C. M.; Adams, P. D.; Adeli, K. et al. Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy 2012, 8, 445-544.
- - please write the abbreviations to the full extent the first time they appear in the text.
Answer: Thank you for your suggestion. We have written the abbreviations to the full extent the first time they appear in the text.
Line 31-32, we added “protein kinase B (PKB/ AKT)”and “AMP-activated protein kinase (AMPK)”.
Line 34, we added “Jun N-terminal kinase (JNK)” and “mitogen-activated protein kinase (MAPK)”
Line 37-38, we added the “peroxisome proliferator-activated receptor gamma (PPARγ)” and the “nuclear factor-kappa B(NF-κB)”.
Line 64, we added the “autophagy-related gene 7 (Atg7)”
Line 71, we added the “HFD”
Line 78, we added “Activating transcription factor 4(Atf4)” and “autophagy-related gene 5(Atf5)”
Line 167, we added the “cholecystokinin-8 (CCK8)”
- - please follow the same order in the material & methods and results sections (That is, in vitro analysis before and then in vivo analysis.
Answer: Thank you for your suggestion. We have swapped the results 7 and 8 in order to make the article more organized
Line 285-385，we exchanged the order of results 7 and 8
Round 2
Reviewer 1 Report
Dear authors,
In page 2 lines 89-91, you mentioned that "we cultured 3T3-L1 preadipocytes.... After the end of the culture,". It is still vague that in figures 1-6, which data was performed with preadipocytes or fully differentiated adipocytes unless you include Oil red O staining and WB or qRT-PCR for essential adipose differentiation genes (e.g. PPARg,CEBPa, UCP1) due to authors' negligence in these critical data. I agreed that you looked at the brown adipose markers in white adipocytes, so it has to be consistent with your mouse data. It is critical that you need to include adipose stem cell isolation experiment from subcutenous depot of mice which has more brown adipocyte trans-differentiation potential. Without these supporting data, it dampens the interest of reviewers. This reviewer strongly recommends authors to include these data in your current paper not in the future investigations. Overall, your research title, aim and focus are not well aligned with your current data and interpretations. In these regards, I strongly recommend you to submit other journals after changing title and research aim. Thanks for kind considerations.
