如果第一次投稿PONE并顺利发表算是“有缘”，那么给它第二次投稿却因作者及评审的原因而未能发表变成了“无缘”。这次投稿一开始就遇到一个“梗”：语言表达能力不行！奇怪了，这篇论文由我执笔，4年前那篇论文也由我执笔，那时就没语言问题？难道PLoS One的编辑策略变了？我推测PONE的要求也许更高了，过去是轻形式重内容，现在则是既重内容也重形式。既然我的英文表达不地道，编辑要求加以改善后送审也在理，这也是编辑对论文质量认真负责不是吗？好吧，那就乖乖拿去润色，先过好第一关再说吧！

稿件润色后再投，编辑决定送审，让我们静候评审结果。过了一段时间，编辑就把两位评审的意见寄过来了。下面就来具体看看他们怎么评价这篇论文吧！在此，我也把我们的简略答复用红色字体附在每道问题的下面，所引用的论文中的字句则用蓝色字体表示：

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Partly

Reply: It could not be possible to confirm all the aspects of a scientific hypothesis in a single manuscript, but we are arranging more supporting experiments for this purpose.

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes
Reviewer #2: No

Reply: A description on Statistical Analysis has been included in the section of Materials and Methods.

3. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes
Reviewer #2: Yes

Reply: Thanks.

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copy edit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.
Reviewer #1: No
Reviewer #2: Yes

Reply: Prior to submission, this manuscript was polished by Editage, but the revised edition was only proofread by ourselves.

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)
Reviewer #1: The topic of this paper is interesting. However,there are some points need to be clarified.
1.There are some spelling mistakes in the paper.

Reply: We have corrected these spelling mistakes as more accurate as possible.

2.The English language of this paper should be revised.

Reply: This manuscript has been revised for improvement of expression by the English language.

3.The "introduction" of this paper is too long. It should be concised.

Reply: The section of Introduction has been shortened, and some implicated description has been moved to the section of Discussion.

4.The author said "90% people had type 2 DM (T2DM), of which 8.3% people were adults". Is this true?

Reply: This sentence involving statistics has been deleted.

5.In this paper, 10^-7,10^-8 and 10^-9 M insulin were used in the experiment. The author needs to explain why these concentrations of insulin were used in the "discussion".

Reply: We wrote in the “Introduction” that “To alternatively intervene with pituitary adenomas, we cultured GH3 cells in either a relatively low glucose (17.49 mM) medium supplemented with insulin (10^-9-10^-7 M) or a relatively high glucose (30 mM) but insulin-free medium. Theoretically, both conditions should induce insulin resistance because 5.5 mM of glucose was defined as a normal glucose blood level (normoglycemia), whereas 25 mM of glucose was categorized as a high glucose blood level (hyperglycemia) [16]. In practical, 10, 15, and 25 mM of glucose and 10^-7 M of insulin were used to induce insulin resistance in 3T3-L1 adipocytes [17] and the hepatoma cell line HepG2 [18], respectively.”

In addition, we also mentioned in the “Discussion” that “We cultured GH3 cells in the medium containing 10^-9-10^-7 M of insulin and 17.49 mM or 30 mM of glucose, which were chosen to induce insulin resistance in adipocytes and hepatoma cells [17, 18]. ”

6.Insulin is suggested to be an important risk factor of some malignancy (such as endometrial cancer, breast cancer and colon cancer). As we know, the biological features of the malignant tumors are different from the benign tumors. Most of the pituitary tumors are benign. The author should explain the association between the results of the current paper and the malignant diseases in the "discussion".

Reply: This manuscript only explores whether insulin is an enhancer of tumor proliferation, but does not engage to answer if insulin is a risk factor of tumor incidence. So we wrote in the “Introduction” that “it should be possible to identify whether insulin would enhance or repress tumor growth rather than cancer incidence. ”

Nevertheless, we thought insulin might be a risk factor of cancer in the insulin-sensitive cells, but is unlikely in the insulin-resistant cells. We wrote in the “Introduction” that “it is also predicted that insulin resistance might decrease the cancer risk even upon insulin administration. In contrast, insulin use in the individuals with insulin sensitivity might increase the cancer risk because of an intracellular high glucose milieu.”

Additionally, we also suggested in the “Introduction” that “it is essential to further investigate the cancer risk after insulin use in the pro-diabetes and DM patients by distinguishing insulin resistance from insulin sensitivity in the near future.” and in the “Discussion” that “Whether insulin sensitivity would increase the cancer risk upon insulin use by activating PI3K-Akt-mTOR signaling and synchronously activating AMPK-SIRT1-PGC-1α signaling merits further elucidation.”

7.The effects of insulin and glucose levels on insulin receptor and IGFBP levels needs to be added.
Reply: Because of difficulty in prepare cell culture in a short time, we plan to determine IR, IGF-1R, and IGFBP levels in our next manuscript.

Reviewer #2: This paper investigates an important issue that has been the subject of many studies. The effects of insulin on pituitary cell function, proliferation and apoptosis are intuitively critical but unresolved.
The authors report studies of H9c2 or RPC cells as controls and rat pituitary GH3 mammosomatotroph cells cultured with insulin at various concentrations(10^-7, 10^-8 and 10^-9M), glucose at high concentrations (30mM) and time courses or 24, 48 and 72 hours. They report effects on cell proliferation, ROS and apoptosis, mRNA and protein levels of Cyclin D1 (CD1) and alpha-fetoprotein (AFP), and mRNA levels of GH and IGF-1.
The preliminary data are interesting but the studies are not sufficiently mature to provide clear answers. The studies require more complete protein data for the insulin receptor and IGF-1, including expression and phosphorylation, as well as interacting proteins, to determine the signaling pathways that are activated and their downstream targets.
Reply: We are going to initiate a series of new experiments to examine more interactive target proteins in the near future, but those data are unable to be collected in this revision because of time limits and technical difficulties.  

Additional minor comments:
The use of H9c2 cells as “normals” is inappropriate and misleading – these are not normal pituitary cells but rather represent myocytes. RPC cells are retinal progenitors. It is unclear why these cells were chosen.
Reply: The term of “normal” has changed to H9c2 or RPC. RPC is the abbreviation of Rat Pituitary Cells rather than that of Retinal Progenitor Cell. H9c2 was chosen because we hoped to exclude any potential effects of GH on our tests, but we found it actually secretes equal GH to GH3 cells, and was replaced by RPC in our next round of experiments.  

The expression of AFP is curious as its expression by adenohypophysial cells is unsubstantiated in the literature.
Reply: Please note our explanation in the “Results” by “although AFP was considered to be mainly synthesized in hepatic cells, it is also expressed in GH3 cells with an almost comparative level to CD1.”

 On the other hand, we also observed a result that “GH was detected from H9c2 cells in a level almost equal to GH3 cells (Figure 4C), whereas IGF-1 was also detected from H9c2 cells in a level higher than GH3 cells (Figure 4D) even though cardiac muscle cells were not considered as the GH-secreting cells or IGF-1-secreting cells. ”

简而言之，两位评委都没有建议退稿的意思，但负面评价占主导地位，尤其是第二位评审，他甚至断言我们使用的RPC细胞是retinal progenitor cell（视网膜前体细胞），后来我们才知道原来他是从Wikipedia中查到的，但我们告诉他RPC是rat puitary cell（大鼠垂体细胞）的缩写。可是，这个辩解不仅没能消除他的疑虑，反而让他开始质疑细胞的真伪。同时，他认为AFP在垂体瘤中表达很奇怪，由此怀疑培养的细胞可能受到污染，这是后话！

第一位评审倒是没有这样的质疑，但要求我们补做更多信号通路实验。对此，我明确表示我们已经安排了新的实验，但不认为应该加在这篇论文中。恰在此时，共同作者中开始出现杂音，担心在PONE上发表论文会败坏自己的名声！不过，我还是力排众议，尽力达成作者、编辑和评阅人之间的妥协，最终希望大修后能顺利发表。为此，我还是补充了一部分新数据，以便让我们提出的科学假说更完善。

这次大修后第一位评审比较满意，只是建议在讨论部分多着点墨就可以接受发表。相反，第二位评审很不满意，认为我们没有改进原有的不足，因此又提出更多更复杂的问题。面对这种情况，我仍不改初衷，再次硬着头皮予以答复。为了避免细胞数据的误导，这次我们删除了“正常细胞”的数据，直接对垂体瘤细胞添加胰岛素前后的结果加以比较。针对他所说未见AFP在垂体瘤中表达的说法，我们也列举了多篇文献证明AFP至少可以在胃癌、肺癌、大肠癌、睾丸癌中表达，而且不一定在肝癌中表达。

Reviewer #1: The authors gave answers to all the comments carefully. If the authors could add some discussion about the association between insulin and malignant diseases, the manuscript will be more wonderful. The paper is suggested to be accepted being published on PloS One.

Reply: We have discussed whether insulin increases or decrease cancer risks depends on insulin sensitivity or insulin resistance because insulin sensitivity allow accelerated glucose uptake and activation of PI3K-Akt-mTOR signaling, but insulin resistance impedes glucose uptake and mimics calorie restriction.

Reviewer #2: The authors have failed to address the criticisms. The most important issues concerning documentation of insulin signaling have been deflected. When they complete their studies, they can submit the paper.

Reply: According to the putative mechanism by which insulin resistance leads to glucose uptake inhibition, we should figure out the reason of insulin resistance. It is well-known that insulin resistance is caused by insulin receptor dysfunction other than insulin receptor deficiency, so the expression levels of insulin receptor and IGF-1 receptor might be not changed, but those receptors  or other downstream constituents might be inactivated by the post-translational modification, such as nitrosylation.

We intended to change our description on the mechanistic link of cancer risks to the conventional insulin/IGF-1 and PI3K-Akt-mTOR signaling because it has been frequently mentioned. To consider an innovation, we suggested a new mechanism context as following:

1. A high-glucose medium simulates hyperglycemia occurring in type 2 diabetes with insulin resistance; 2. Insulin resistance inhibits glucose uptake and leads to cell hungry; 3. Cell hungry mimics calorie restriction to downregulate IGF-1 and NOS2/ iNOS; 4. Downregulation of IGF-1 and NOS2/iNOS exert an anti-tumor effect by rectifying inflammation-induced mitochondrial dysfunctions. To confirm those correlations, we added the new experimental data derived from NOS2 knockout, which not only upregulates anti-oxidation responsible genes, including FOXO3a, HO-1, EPO and VEGF, but also downregulates the oncogene cyclin D1 and upregulates the tumor suppressor gene p21.

The quantification result from IGF-1 downregulation was still included because our suggested CR-like manner also downregulates IGF-1. Detection of IGF-1 in pituitary adenoma should not be from contamination because both IGF-1 mRNA and protein are fluctuated synchronously. Additionally, a non- specific expression of the tissue-specific IGF-1 in tumor cells was commonly observed, such as in melanoma-initiating cells and multiple myoloma cells, so it was not surprised that IGF-1 expressed in the liver can be also expressed in pituitary adenoma cells, and occasionally it is very highly expressed, as seen in this study.

The minor comments were treated poorly

1. "normal" remains in the paper and figures.

To avoid misleading, we deleted all data from “normal” cells (H9c2 and RPC) because it can be adequate and sufficient to compare untreated tumor cells with treated tumor cells.

2. The use of myocytes is inappropriate and the suggestion that they secrete GH suggests contamination, since this has not been reported elsewhere.

The data from H9c2 myocytes were no more used to compare with untreated and treated tumor cells in this revised manuscript.

3. If the authors have created their own normal pituitary cell line and called it by the same name as a retinal progenitor cell line, they must validate their cells.

The data from RPC pituitary cells were also deleted in the revision.

4. The production of AFP by GH3 cells also suggests contamination.

Although AFP is highly expressed in hepatocellular carcinoma, it can be also detected from other types of tumor, for example, gastric cancer, testicular neoplasm, lung squamous carcinoma, and colorectal cancer, as described in the following incompletely cited references.

Clinicopathologic and prognostic characteristics of alpha-fetoprotein-producing gastric cancer.

He R, Yang Q, Dong X, Wang Y, Zhang W, Shen L, Zhang Z. Oncotarget. 2017 Apr 4;8(14):23817-23830. doi: 10.18632/oncotarget.15909.

Mildly elevated serum alpha-fetoprotein (AFP) among patients with testicular cancer may not be associated with residual cancer or need for treatment.

Wymer KM, Daneshmand S, Pierorazio PM, Pearce SM, Harris KT, Eggener SE.

Ann Oncol. 2017 Apr 1;28(4):899-902. doi: 10.1093/annonc/mdx012.

AFP-producing lung squamous carcinoma.

Liu M, Liu B, Zhou Y, Quan X, Liu B, Dong L.

QJM. 2016 Dec;109(12):813-814. doi: 10.1093/qjmed/hcw179. Epub 2016 Nov 8.

Diagnostic Performance of a Novel Multiplex Immunoassay in Colorectal Cancer.

Dressen K, Hermann N, Manekeller S, Walgenbach-Bruenagel G, Schildberg FA, Hettwer K, Uhlig S, Kalff JC, Hartmann G, Holdenrieder S. Anticancer Res. 2017 May;37(5):2477-2486.

Additionally, not all hepatocellular carcinoma expresses AFP, as cited below.

Diagnosis of AFP-negative early-stage hepatocellular carcinoma using Fuc-PON1.

Shu H, Li W, Shang S, Qin X, Zhang S, Liu Y. Discov Med. 2017 Mar;23(126):163-168.

既然第二位评审者的评价如此负面，编辑只好要求我们再次修改。可是，由于这位评审者坚持认为细胞受到污染，已经接近全盘否定已有的实验结果，甚至流露出对数据的怀疑，乃至对人品的不信任，我开始有些失去耐心。这时，编辑本着负责任的态度，又邀请了第三位评审人。下面就来看看这次他们是如何评价的，而正是这位新评审的加入，让我们彻底断绝了在PONE发表此文的念头！

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: (No Response)

Reviewer #3: (No Response)

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #2: No

Reviewer #3: No

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: N/A

Reviewer #3: I Don't Know

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #2: Yes

Reviewer #3: Yes

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #2: Yes

Reviewer #3: No

6. Review Comments to the Author

Reviewer #2: The authors have not addressed the concerns raised in the initial review. There are serious concerns about the cell lines. The authors seem to have created their own "RPC" cells but these have not been validated. The expression of GH by cardiac myocytes is unprecedented and suggests cell line contamination. Similarly the expression of AFP by pituitary cells indicates contamination.

The failure to address concerns about insulin receptor and IGF-1 signaling is a fatal flaw in this manuscript.

Reviewer #3: In this manuscript, the authors attempt to clarify the mechanisms governing insulin-dependent cancer development observed in large epidemiological studies. The rat pituitary adenoma cell line GH3 is stimulated by different concentrations of insulin and/or different levels of glucose, and the expression of tumor markers as well as cell proliferation, oxidative stress and apoptosis are observed.

There are many concerns with this study.

Major concerns:

1. While extensive experiments have been conducted, the experimental approach as well as the manuscript lacks comprehensibility. The manuscript is extremely difficult to read; the flow of logic is frequently not clear and the language is often inaccurate or faulty. The manuscript would require thorough revision, with the help of a native English speaker, for language and logic. Concerning content, the manuscript and especially the discussion appear as if pieced together in a highly data driven approach.

2. If and how exogenous insulin contributes to cancer development is a very relevant question, especially considering the ever increasing prevalence of diabetes mellitus under insulin therapy. However, the experimental system used in this study is poorly suited to explore this problem:

- Pituitary tumors, while relatively common incidental findings, have not been associated with insulin use in large epidemiological studies.

- Pituitary tumors are benign and well differentiated tumors with very infrequent malignant transformation. As such, GH3 cells do not represent an adequate general model for potentially insulin-dependent tumors.

- In pituitary somatotropes and GH3 cells, insulin and IGF-1 specifically act to inhibit GH production and release as a part of the negative feedback mechanism within the GH/IGF-1 axis; the intracellular mechanism governing this specific insulin and IGF-1 effect is unlikely to be relevant in other tissues.

3. Fig 3 CDE: the middle (48h) panel of AFP appears to originate from a different blot with a smoothed edge. If this is indeed the case, this modification would qualify as an inappropriate modification as specified by PLoS one author guidelines.

4. No adequate explanation is provided for the expression of IGF-1 or AFP by pituitary somatotropes. While it is possible that this is indeed the case, contamination remains a distinct possibility.

5. Authors state that they used insulin-free medium for experiments with high glucose and without insulin. They suggest that the discrepancies between mRNA and protein expression of AFP and CD1 are due to cellular starvation in both “high” and “higher” glucose states – this is reflected in the manuscript title and the abstract. Normal medium containing fetal bovine serum, as described in the methods section, contains insulin that is sufficient for maintenance and adequate glucose uptake of most cell lines including GH3 cells without additional insulin. What is the evidence for cellular starvation and a “calorie-restriction” like state of those cells? Furthermore, if the glucose added to the medium cannot be taken up by the cells, how do you explain the differences observed between “high” and “higher” glucose?

Minor points

1. GH secretion is significantly affected by insulin at 10^-9 M while the effects on proliferation are only seen at an insulin concentration of 10^-7 M, suggesting that the proliferative effect may only be evident at supraphysiological doses.

2. Fig 2A: Significances refer to what?

3. Fig 2CDE: “ATP” should probably read “AFP”

4. What is meant by a “non-specific expression pattern” of AFP in GH3 cells?

5. How do you explain the discordant results concerning CD1 and AFP expression in response to insulin versus glucose? Patients with DM would be expected to exhibit both hyperglycemia and hyperinsulinemia.

6. The standard medium used in the experiments (17.49 mM glucose) already contains high levels of glucose comparable to hyperglycemic levels in a diabetic individual. What is the rationale for the two different high glucose concentrations? A control simulating normoglycemic conditions should be provided.

7. Figure numbers given in the results section are incorrect.

8. Data in Fig 4 and Fig 5 should be presented in a more comprehensive way.

9. The immediate relevance of the final piece of the results section (Fig 6C and 6D) is unclear. These appear to have been derived from a different hypothesis and should probably be removed.

10. Methods: The method for Western blotting and FACS (“according to manufacturer’s manuals”) is unclear.

11. Methods: The number of replicates for each experiment is unclear.

12. The statistical analysis is unclear: were adjustments performed to account for multiple comparisons?

这位新评审者说：培养基中加有胎牛血清，应有足够的胰岛素，由此断定我们所谓insulin-free的实验条件不成立！这真是“天方夜谭”啊，即使胎牛血清中有残留胰岛素，但也是经过热处理的，他为什么就没看到呢？为了更准确起见，我还专门请教了有关专家并咨询供应商，他们都没有听说过培养基中含有胰岛素的说法。

尽管编辑尽职尽责，要求我们再次修改，可是让我们从何下手呢？经过征询共同作者的意见，大家一致同意撤稿，决定先在bioRxiv上占个先，待新数据出来后重新向其他刊物投稿。

总之，这次论文发表不成功完全不能怪PONE，也与编辑没有半毛钱关系，我认为他是尽职尽责的，自始至终无可指责，应该是为PONE加分的，为他点个赞！反而我们有些自责与内疚，浪费了编辑和评审那么多时间和精力，结果却“一言不合”就撤稿，PONE并没有从中赚取任何经济利益，反而贴了不少“冤枉钱”。最后我想弱弱地问你：你还认为PONE“唯利是图”吗？编辑“放水”吗？评审“合谋”吗？