Dear Editor,
We have studied the valuable comments from you, the assistant editor and reviewers carefully, and tried our best to revise the manuscript. The point to point responds to the reviewer’s comments are listed as following:
Responds to the rev iewer’s comments:
Reviewer 1
Comment 1: in page 3, line 40, we " changed to rats were Response: According to the reviewer’s comment, we have corrected the sentence. Furthermore, we have had the manuscript polished with a professional assistance in writing.
Comment 2:page 25. The style of reference 40 is not right (using initials for the first names). Since this paper has been published, the volume and page Nos should be provided.
Response: Thank you for your careful work. We have added the volume and page numbers for reference 40.
Reviewer 2
Comment: I would like to thank the authors for their efforts in addressing the criticisms with additional experiments. The one criticism that they did not address was relating to energy expenditure as the reason that the animals on the low calcium diet gained more weight. While I understand that performing this experiment will not affect the conclusion of this manuscript, I do believe that this point could be discussed in the Discussion section.
Response: Thank you for your valuable advice. Based on the previous revision, we further address the relationship between low calcium diet and energy expenditure in the section of discussion according to your thoughtful comments.
Reviewer 3
Comment 1: In the text you often write: “As previously described”. Unless that paper is from your lab or one of the method paper co-authors is on the present MS this is not quite proper since the statement infers method development from your lab. There are numerous instances like that in the methods section; these should all be changed “according to those described by…..”
Response: We are sorry for this language mistake. We have carefully corrected this phrase throughout the manuscript according to your comment.
Comment 2: There are still some wording, sentence structure and grammatical issues even in this basically well put together MS. For example, while authors may have been excited about the data you cannot start a sentence with “Excitedly” in line 418 or “Whatever” in line 395.
Response: Thank you very much to point out the sentence structure and grammatical issues in our manuscript. According to the comments from you and the editors, we polished the manuscript with a professional assistance in writing, conscientiously.
Comment 3:In my view a big omission in this work is ignoring the anabolic side of lipid metabolism as well as thermogenesis issues. For example all animals consumed the same amount of feed but we had extra fat storage in the low Ca diet groups. So where did the extra energy go? Zemel et al (citation 34) in similar work indicate that increased thermogenesis on the high Ca diet explains the dissipation of dietary energy. Further even though Zemel et al (#34) indicated lipogenesis was enhanced in the low Ca diets that was in 2000 and you should have monitored expression of FAS and UCP either as mRNA abundance or actual FAS/UCP changes via proteomics or blotting techniques. In any case these controls are missing here and not emphasized in the MS. Casual reading of this paper would lead to the conclusion that the dietary Ca effect on fat deposition is strictly a function of increased or decreased lipolysis. While lipolysis appears to be a major player, lipogenesis and thermogenesis cann
ot be ignored for completeness. In Fig 8 you also show a decline in cAMP for the low Ca diet. Well beta agonists or cAMP enhancers regulate transcription of adipose and liver FAS (in rats (J Biol Chem 271:2307, 1996) and recently with large animal models (Hausman et al J Animal Science 87:1218, 2009 and Halsey et al J Animal Science 89: 1011, 2011). In addition
cAMP levels could have been monitored. I really do not like the last sentence in the Abstract line 47-50 where you state that “low calcium diet-induced increase in fat mass was due to enhanced lipogenesis mediated by an upregulated CaSR signaling pathway” Your results here show no such thing, this is a completely false statement based on data herein. Correct. You show that high Ca diets enhance lipolysis and low Ca diets are antilipolytic. You did not monitor lipid anabolism here at all. See also line 255-257 and lines 333-335 of your MS. Response: Thank you for your valuable and thoughtful comments. As you suggested that the anabolic side of lipid metabolism as well as thermogenesis issues should be monitored. We really agree with your viewpoints. In the present study, we did find that low calcium diet increased the mRNA level of fatty acid synthase (FAS) in white adipose tissue. Furthermore, the FAS mRNA level were also increased in adipocytes after treatment with 1,25-(OH)2D3in in-vitro experiments. However, the increased FAS mRNA levels were not affected by preventing either the nuclear vitamin D receptor (nVDR) or calcium-sensing receptor (CaSR), suggesti
ng that FAS might not be involved in the CaSR pathway. In addition, we thought that FAS played its role in fatty acid synthesis mainly in liver previously. Besides, the manuscript was required to restrict number of total words and our previous focus was on the antilolytic role of CaSR in the process of fat accumulation. So we ignored to provide the data of FAS mRNA levels in the submitted manuscript. In the newly submitted manuscript, we have provided the mRNA levels according to your helpful suggestion.
We have reported the effects of dietary calcium on UCP2 mRNA levels in adipose tissue and UCP3 in skeletal muscle in our previous studies (1, 2). Thus, we believed that low calcium diet led to decreased thermogenesis in the present study. It was a pity that we did not measure the rat core temperature in those studies. The UCP2 mRNA levels in adipocytes were observed to be decreased after treatment of 1,25-(OH)2D3. This effect was prevented by using nVDR CaSR gene silencing but not by CaSR gene knockdown, suggesting that UCP2 was not involved in CaSR pathways. In the newly submitted manuscript, we have provided the UCP2 results.
Thank you for your careful reading of our manuscript. We are very sorry for our fault statement in the abstract. We have corrected it in the new manuscript.
editor版本Comment 4: A point that does not emerge well from the discussion is how low Ca intakes result in higher intracellular [Ca] concentrations and really the effects on fat
deposition in the cells in many ways are due to an increased intracellular Ca level mediated via CaSR expression increases and the effect of VitD3 on nVDR show in Fig 8. The authors must remind readers that Ca levels in the blood are under hormonal regulation (Calcitonin, PTH and VitD3). Thus when diets low in Ca are consumed and blood Ca decline, PTH and VitD3 are called upon to mobilize bone Ca to replenish the blood Ca. Then coupled with an increase in CaSR more Ca actually is found in AT despite the fact that many would think the AT Ca level should decline. The reason is that tissue/circulating Ca levels are not diet depended but regulated. The vast bone stores of Ca will provide ample Ca here especially during a study of this length. While authors address these issues maybe could be presented in a less complicated discussion.
Response:Thank you for your instructive suggestions. We are sorry for not describing the effect of low calcium diet on intracellular calcium concentrations mediated by CaSR, as well as the impact of hormone regulation on serum calcium levels clearly. According to your helpful advice, we have rewritten these two parts in the section of discussion. Thank you again.
Comment 5: Not all citations are in JN style
Response: We have careful recheck and corrected the style of the citations according to the requirement of JN.
Comment 6: Abstract conclusion differs from lines 255-257 and 333-335; WHY? Response: Thank you for your careful reading of our manuscript. The conclusion from lines 255-257 is about the effect of low calcium diet on serum levels of free fatty acids (FFAs) and lipids. We considered FFA and glycerol as indicators of TG hydrolysis in adipose tissue. The low calcium diet caused decreased serum FFA and glycerol levels without influencing lipoprotein lipase (LPL) activity, so we thought the lipolytic effect of adipose tissue to be suppressed by low calcium diet. The conclusion from lines 333-335 was about the effect of 1,25-(OH)2D3 whose levels were increased under low calcium conditions on lipolysis. We used the glycerol level as the indicator of TG hydrolysis in adipocytes. Both the in vivo and in vitro experiments showed low calcium status caused an antilipolytic effect.
Comment 7: Line 150-153. The qRT-PCR methodology is not at all understandable as you cite a Texas A&M published paper. This is completely insufficient with the newly established standards on gene expression via qRT-PCR. There is no mention of efficiencies of amplifications in these data nor how the use of the reference gene was established etc. I think Pfaffl and Bustin have recently written an article on this; please totally revise 150-153 in line with what you did and applying the new standards.
Response: Thank you very much. Because the JN restricts the number of total words of manuscript, we cited the Texas A&M published paper. In the newly submitted manuscript, we describe the detailed protocols in our lab.
Comment 8:Line 179 on Not clear as in sentences talk about different AT cell vise.
Response: We are sorry for not addressing the adipose tissue cell sources clearly. We have rewritten the methods.
Comment 9: Any previous documentable work with siRNA?
Response: Yes, we have documentable work with siRNA in our research team. The results were published in the journal of Biochem Biophys Res Commun (3).
Comment 10: Line 214.. Cultured primary rat adipocytes and SW872 adipocytes ……
Response: Thank you very much. According to your comment, we have had the manuscript polished and corrected the mistakes.

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