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    Reply to the reviewers

    Manuscript number: RC-2021-01219

    Corresponding author(s): Rajan, Akhila

    1) General Statements [optional]

    This section is optional. Insert here any general statements you wish to make about the goal of the study or about the reviews.

    The goal of this study is to:

    • Define how prolonged exposure to a high-sugar diet (HSD) regime alters both the lipid landscape and feeding behavior.
    • Determine how changes in lipid classes within the adipose tissue regulates feeding behavior. Key findings:

    In this study, by taking an unbiased systems level and genetic approach, we reveal that phospholipid status of the fat tissue controls global satiety sensing.

    Impact of Key findings:

    By uncovering a critical role for adipose tissue phospholipid balance as a key regulator of organismal feeding, our work raises the possibility that the rate-limiting enzymes in phospholipid synthesis, including Pect, are potential targets for therapeutic interventions for obesity and feeding disorders.

    Peer review comments:

    This study has immensely benefited from the thoughtful peer-review of three reviewers. As per their recommendations, we have performed a major revision by performing additional experiments (see summary table below in next section) and strived to address the major concerns raised. Based on our reading, there were two major concerns that overlapped between all three reviewers raised. They are as follows:

    • Does the genetic disruption of Pect in fly fat body alter phospholipid levels? Two reviewers (#2 and #3) recommended that we perform lipidomic analyses on adult flies with adipose tissue specific knockdown of For the revised version, we have completed this lipidomic experiment, and present results as a new main Figure 6, Supplemental S7 and S9.
    • Is the dampened HSD induced hunger-driven feeding (HDF) behavior because of increased baseline feeding (#1 and #3)? In addition, reviewer #1, asked us whether HSD flies experience an energy-deficit? In other words, we were asked to uncouple whether what we observed was HSD-driven allostasis or indeed, as we had interpreted, that HSD dampened hunger-driven feeding response.

    Hence, they recommended that we:

    1. Re-analyze our hunger-driven feeding datasets and present non-normalized data (also requested by Reviewer #3) and show baseline feeding behavior on HSD. To address this, we have completed this analysis and present our results in Figure 1B-D and S1.
    2. Determine whether the HSD fed flies display an energy deficit on starvation. To this end, we performed an assayed starvation-induced fat mobilization on HSD, results for this are now presented on Figure 1E-G and S2. Conclusions after the revision:

    First, it is important to note here that the additional experiments have not caused a significant revision of the major conclusions of the original version of our study. In fact, we hope that the revised version provides clarity and further substantiation to our original arguments.

    • The lipidomics experiments on Pect fat-specific knock-down flies show that reducing Pect in fat-body causes a significant reduction in certain PE lipid species (PE 36.2 specifically- Figure 6B). This is consistent with a prior report on lipidomics of the Pect null allele by Tom Clandinin’s group (PMID: 30737130). Furthermore, we note that when Pect is knocked down in the fat body, there is a significant increase in two other classes of phospholipids LPC and LPE (Figure 6A). Together, this suggests that an imbalance in phospholipid composition in the absence of Pect activity in fat.
    • The starvation-induced fat mobilization experiments show that despite being fed a prolonged HSD, adult flies sense starvation and effectively mobilize fat stores, at a level comparable to Normal food (NF) fed adult flies, suggesting that even despite HSD exposure, adult flies experience an energy deficit on starvation.
    • In our non-normalized data, we find that the baseline feeding events are not significantly altered between HSD and NF-fed flies (Figure 1D). This suggests that the effects we observe are not due to an increase in the “denominator”, but a dampening of hunger-driven feeding on HSD. With regard to our original version, all three peer-reviewers found that the study was interesting, significant, important, and novel – Reviewer #1: “The work is potentially novel and interesting”; #2 : “I find the study to be potentially very important - the authors combine a longitudinal study that would be difficult in any other model with the powerful genetic tools available in the fly. The conclusions are mostly convincing”; #3: “This manuscript demonstrates how fat body Pect levels affect HSD induced changes in hunger-driven feeding response. I agree with all the reviewers points; potentially very interesting”. But had requested that we provide further substantiation and clarification.

    We sincerely hope that the peer-reviewers find that our revised version with additional new experimental datasets, improved data visualization, and the presentation of non-normalized raw data points, makes this study clear, compelling, and well-substantiated.

    • Point-by-point description of the revisions This section is mandatory. *Please insert a point-by-point reply describing the revisions that were already carried out and included in the transferred manuscript. *

    Below we summarize in Part A, the key experiments that were performed to address the major concerns. In Part B, we provide a point-point response to each reviewer with embedded datasets.

    Part a:

    We performed several new experiments, including:

    • To address the primary concern of Reviewer #1 regarding whether the HSD flies have a similar energy deficit to Normal food (NF) fed flies, we performed analysis of stored neutral fat Triacylglycerol (TAG) reserves and how HSD fed flies mobilized fat stores on starvation. We present these results in Figure 1E-G, S2. These results show that HSD-flies despite accumulating more TAG (S2), breakdown a similar amount of fat reserves as NF-fed flies on starvation at any time-point (Figure 1E-G). This suggests that HSD-fed flies do sense and respond to energy deficit.
    • To address concerns of reviewer #2 and #3 on whether Pect genetic manipulation affects specific phospholipid classes, we performed lipidomic analyses. The table below summarizes the new 3 new figures and 4 supplemental figures (blue text are all new figure numbers and figure panels) and three new Supplementary files as per reviewer’s request.

    Figure #

    Main point

    New datasets in revision

    Companion Supplement

    1

    HSD alters feeding behavior, but flies still breakdown TAG on starvation.

    TAG storage and breakdown over longitudinal HSD shows that HSD and NF fed flies show similar levels of TAG breakdown on starvation, despite consistently elevated TAG on HSD. This supports the idea that flies do sense starvation even on HSD, but there is a uncoupling of the feeding behavior after Day 14. Revised the data representation of Figure 1 to show non-normalized data over time. S1 and S2 companions are new in the revision. Panels 1D to 1E are new for the revision.

    S1- Raw data of feeding events plotted.

    S2 Elevated TAG at all time points.

    2

    HSD causes insulin resistance

    S3A added to show that insulin transcript levels remain the same in response to reviewer #3’s concerns.

    S3

    3

    Phospholipid concentration raw data from lipidomic on Day 7 and Day 14 HSD suggest that PC, PE levels are increased on Day 14 HSD.

    Figure 3 revamped to show new data visualization and non-normalized raw data to address Reviewer #2’s major concerns. S4A and S4B added. In addition Supplementary File 1 and 2 provided with raw lipidomics data as per reviewer #2’s request.

    S4.

    S4A- non normalized raw data of all other lipid classes on HSD.

    S4B- fatty acid species data on Day 14 added as per request of rev.#2.

    4

    HSD regulate Apo-I levels in the IPCs and phenocopies Pect KD.

    Added Figure 4A to show that HSD phenocopies Pect-KD in terms of delivery to brain

    S5 showing the validation of the Apo-I antibody.

    S6 validation of Pect KD and over-expression and Pect mRNA levels dysregulation on HSD.

    5

    Pect RNAi is insulin resistant

    N/A

    N/A

    6

    Pect knockdown shows significant increase in LPC and LPE, and a non-significant reduction in PC, PE levels. Specifically, the PE lipid class PE36.2 is downregulated.

    Fig 6, S7, S9 are completely new based on reviewer #2 and #3 requests. In addition Supplementary File 3 provided with raw lipidomics data as per reviewer #2’s request

    S7, S8, S9#.

    S7- new Pect KD other classes

    S8- new PE classes for day 14 and Pect associated classes.

    S9- Pect OE lipidomics

    7

    Pisd and Pect activity in adipocytes are required for hunger-driven feeding behavior in normal diets

    Pisd RNAi data was moved from supplement to main figure.

    N/A

    Note on revised text: We have revised text not only in the results section, but also as per reviewer #2’s recommendation, we have revamped our introduction and discussion as well. Since the manuscript has been significantly revised to include a main figure 6, fully altered Figure 1 and 3, multiple new supplemental figures, the changes in text are extensive. Hence, they are unmarked in the main text. Nonetheless, we hope that the reviewers will be able to evaluate these changes, as we have provided the specific locations in text and embed key figures in the point-point response below.

    __Part B: __Point-Point responses to reviewer comments.

    Reviewer #1 comments in Blue, author response in black.

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    In this manuscript, Kelly et al. show that the difference between the feeding behavior of fed and starved flies (hunger-driven feeding; HDF) is absent in animals fed a high-sugar diet (HSD) for two weeks or more. The disappearance of HDF with HSD coincides with changes in phospholipid profiles caused by HSD. Furthermore, RNAi-mediated downregulation of Pect in the fat body-a key enzyme in the PE biosynthesis pathway-phenocopies physiological effects of HSD. Moreover, downregulation or overexpression in the fat body abolishes or induces HDF, respectively, abolishes or induces HDF, respectively, independent of HSD treatment.

    Overall, the manuscript is well-written and the phenotypes are clear. However, I have major concerns regarding the authors' interpretation of the data and their conclusion. Most importantly, while it is clear that the authors' high-sugar dietary treatment affects feeding behavior and physiology, I am not convinced that the changes can be considered "hunger-driven"-which is central to the main point of the manuscript. Therefore, it is my recommendation that the authors substantially revise the manuscript by either showing additional/re-analyzed data that rule out alternative hypotheses, or rewriting the manuscript keeping alternative interpretations in mind.

    We are thankful to this reviewer for their thoughtful critique, and constructive and specific suggestions on how we can redress these concerns. We have taken on board the concerns of this reviewer regarding our interpretation of whether the changes in feeding behavior can be considered hunger-driven or not. Based on their advice, we have made significant changes by addressing: i) does HSD increased baseline feeding- we now show non-normalized raw data and data supports conclusion that baseline feeding is not higher; ii) whether HSD- fed flies can sense an energy deficit at levels similar to NF fed flies- we show that HSD flies sense energy deficit. We have provided detailed response below, and we hope the reviewer finds the additional datasets and re-analyzed data are consistent with the interpretation that prolonged HSD dampens starvation induced feeding. In addition to this key concern this reviewer has made a many other salient points that we have addressed with additional data or by clarifying the text.

    Major comments:

    1. The data do not sufficiently show that the long-term HSD regime disrupts "hunger-sensing." The manuscript should address alternative hypotheses by showing raw instead of normalized data, rewriting the manuscript with a new central conclusion, or running additional experiments that actually show a defect in hunger-driven response. a. The main results that the authors rely on for the argument is that the ratio of feeding events that the starved and non-starved flies eat is different between the groups fed normal or HSD. However, because the authors only show normalized data (normalized to non-starved flies; Fig. 1), it is difficult to tell whether the change is due to a chronically increased feeding in non-starved HSD flies-maybe in perpetual hunger-like allostasis-or dampened starvation response. Indeed, the data shown in Fig S1 show that flies fed HSD for as short as 5 days show more frequent feeding events compared to age-matched controls fed normal food. It is possible that because the HSD-fed flies eat more than NF-fed flies, even without being starved, the ratio of starved/non-starved feeding is lower in the HSD-fed group-due to changes in the denominator, rather than the numerator.

    We have taken onboard this concern regarding presenting only normalized data, and that clouded the interpretation and left open other possibilities. In the completely revised figure 1 and S1. We now show non-normalized data, as a function of time. First we note that HSD-fed flies, do not show higher baseline feeding that NF fed flies, except on Day 10 of HSD, when there is a modest but significant elevation (Figure 1D).

    Nonetheless, on Day 10 HSD, flies still display increased hunger-driven feeding HDF (Figure 1C), it is only after Day 14 HSD that HSD dampens the starvation induced feeding.

    1. It is also possible that the HSD-fed flies are simply not in as big an energy deficit physiologically, due to the increased fat deposits they've accumulated (as the authors show later in the manuscript). It may take longer for the fat HSD flies to reach substantial energy deficiency than the NF flies, but they still may eventually be able to appropriately respond to hunger, just like NF flies. In such case, it would be a misnomer to call this behavioral change a 'defect in hunger-driven feeding behavior.' Maybe an experiment with a dose-response curve of "hunger driven feeding response" as a function of duration of starvation would help? Prompted by this reviewers question, we asked whether HSD fed flies, that have a higher baseline neutral fat store (Triacylglycerol-TAG) level, and if HSD-fed flies can sense energy deficit. For this, we revisited the longitudinal assays for neutral fat triacylglycerol (TAG) storage that our lab had generated, along with the HSD-HDF studies. We now present this evidence as Figure 1E-1G and Figure S2. Overall, our experiments point to the idea that adult flies fed HSD, are able to sense and mobilize TAG stores effectively throughout the 28-day time point that we analysed.

    First as shown in Figure S2, flies fed HSD display an increase in TAG levels. But it is to be noted that while TAG stores increase, the increase is not linear with time. This suggests that adult flies exposed to HSD store excess energy as TAG, but the increased TAG stores stay within a certain range despite the length of HSD exposure. This suggests that adult flies on HSD still display TAG homeostasis.

    Next, to directly address the reviewers point about HSD fed flies not sensing an energy deficit, we subject HSD-fed flies to an overnight starvation, same regime as used in the overnight feeding experiments, and asked whether they mobilize TAG. We noted that flies exposed to HSD breakdown TAG throughout the 28-day exposure at statistically significant levels for Day 3- Day 28, except on 14 and 21 days (Figure 1F). While there is TAG mobilization on Day 14 and 21, the difference is not statistically significant. Nonetheless, we note the same levels TAG breakdown for normal lab food (NF) fed flies on Day 14 and 21 (Figure 1E). Overall, HSD fed flies sense and display energy deficit, as measured by TAG store mobilization, throughout the 28 days of HSD exposure, at levels comparable to NF-fed flies (Figure 1G).

    Taken together, these results suggest that while HSD-fed flies experience an energy deficit on starvation, at levels comparable to NF-fed flies, throughout the 28-day time point assayed. But, their starvation driven feeding-response is dampened by Day 14 and by Day 28, the HSD-fed flies display more feeding events than HSD starved flies. These results are consistent with the interpretation that in HSD-fed flies the starvation-induced feeding behavior becomes desynchronized from the starvation induced TAG-mobilization, suggesting that there is an absence of hunger-driven feeding.

    1. How can you be sure that lower Dilp5 immunofluorescence is indicative of increased Dilp5 secretion? Wouldn't decreased production of dilp5 also have the same results?

    It has been shown previously in HSD fed larvae are hyperinsulinemic, i.e., they have 55% increase in circulating Dilp2 ( PMID: 22567167). Additionally, we have shown that ectopic activation of the insulin-producing neurons by expressing TRPA1, an ion channel that activates neurons, reduces Dilp5 accumulation without a change in Dilp5 mRNA levels (PMID: 32976758), suggesting that reduced Dilp5 accumulation, without alterations to mRNA levels is a proxy for increased secretion. Now, in response to this concern, in the revised manuscript, we have added qPCR data of Dilp2 and 5 (Figure S3A), which show no difference in expression levels after 14 days on HSD. Therefore, there is no dip in Dilp5 mRNA production. Given that Dilp2 and Dilp5 mRNA levels remain the same, but we see reduced Dilp5 accumulation, we interpret this to mean that Dilp5 secretion is increased.

    1. Also, the authors should state in the main text that it is Dilp5, not just any Dilp. Thanks for this suggestion and we have fixed this and referred to Dilp5 specifically throughout the text in the results section.
    1. Data presentation: a. Sometimes the data are normalized to NF (Fig 4B-C), sometimes not (ex. Fig 4A, S4C). Unless there is a specific rationale for the data transformation, it would be more appropriate to show untransformed data (ex. Fig 4A, S4C), especially as the authors use two-way ANOVA to determine significance. Only showing the differences implies comparison against a hypothetical mean (i.e. μ0=0), not between two group means.

    We thank the reviewers for bringing this issue to our attention. We updated all the figures to show untransformed data in the revised manuscript.

    Some figures show both individual data points and summary statistics (mean, SD, ... ex. Fig 2A)-which I believe is ideal-but some show only one or the other (ex. Fig 2B, no summary statistics; Fig. 3, no data points. The manuscript would read more convincing if data visualization is consistent across figures. We thank the reviewers for their feedback. We have made changes to all the figures in the revised manuscript to improve visual consistency.

    Minor comments:

    1. High sugar diet: what is the actual sugar concentration in the NF v. HSD diets? The authors write that the HSD diet contains "30% more sugar" than the NF, but providing the final sugar concentrations-sucrose or others-would be informative for other scientists studying the effect of high sugar diets.

    We thank the reviewer for their suggestion and now we have updated the methods to include this sentence. After 7 days, flies were either maintained on normal diet or moved to a high sugar diet (HSD), composed of the same composition as normal diet but with an additional 300g of sucrose per liter”.

    1. Additionally, the definition of HSD is inconsistent. Main text (Page 5, line 17) states that their HSD is "60% more sugar than normal media," whereas the figure legend (Fig 1) and the Methods state that the HSD contains "30% more sugar." We apologize for this egregious typo in the figure legend! We have now fixed this to say 30% HSD. Only 30% HSD was used throughout this study.
    1. Starvation medium: please provide justification for why the authors used 1% sucrose/agar for starvation medium, instead of plain agar/water that most labs use. At least clarify and provide a reference for the claim that the 1% sucrose/agar "is a minimal food media to elicit a starvation response."

    We are very grateful for this reviewer identifying this this methods description error and bring it to our attention. We used 0% sucrose agar for overnight starvation in this study as most labs do. The error occurred because we were using another manuscript from the lab to help draft the methods section (PMID: 29017032). In that study, where we assayed the effect of chronic starvation our lab used: “1% sucrose agar for 5 days at 25C”. However, in this current study, because we are testing acute effects of overnight starvation, we are using 0% sucrose agar.

    1. Pect mRNA level is higher with HSD. This is surprising because not only, as authors mention, is increased PC32.2 with HSD suggests lower Pect activity, but also because Pect RNAi phenocopies long-term HSD in HDF behavior, lipid morphology, FOXO accumulation in fat body. The authors speculate that the data "likely shown an upregulation in an attempt to mediate the Pect dysregulation occurring at the protein level." If that were true, a western blot may be informative. Zhao and Wang (2020, PLoS Genetics) generated a Pect antibody that seems compatible with western blot applications. That being said, I don't think such data is critical for the manuscript. I mention this simply as a suggestion for the authors. a. page 8, line 22-23, did you mean to write "Given how PC32.2 is elevated after 14 days of exposure to HSD, we assumed that Pect levels would be low for flies under HSD," not "high?" Otherwise the subsequent 2 sentences don't make sense.

    We agree that the most confusing aspect of the study was that Pect mRNA levels being very high on Day 14 HSD, but nonetheless the effects of Pect-KD phenocopied HSD. To resolve this, we have now performed lipidomic analyses on whole adult flies, when Pect is knocked-down (KD) by RNAi in the fat tissue. We now present a new dataset in Figure 6. Two striking changes occur. They are:

    1. Pect-KD shows increase in the phospholipid classes LPC and LPE (Figure 6A). In contrast, LPE is significantly downregulated on HSD Day 14 (Figure 3).
    2. Pect-KD shows a significant reduction in specific class of PE 36.2 (Figure 6B). Our data regarding increase in PE 36.2 agree with a previous lipidomic analyses of Pect mutant retina (PMID: 30737130). In contrast, PE 36.2 trends upwards on 14 day HSD (Figure S7C) though not significantly. On 14-day HSD consistent with extreme upregulation of Pect mRNA fed flies (Figure S6A; Pect mRNA 200-250 fold), PE trends upwards on 14-day HSD (Figure 3) and PE 36.2 trends higher (Figure S7C). We note that on the surface of it PE and LPE per se are contrasting between 14-day HSD lipidome and fat-specifc Pect-KD. But there is a significant commonality that under both states there is an imbalance of phospholipids classes PE and LPE. Hence, we propose that maintaining the compositional balance of phospholipid classes PE and LPE is critical to hunger-driven feeding and insulin sensitivity. Hence, either increase or decrease, of these key phospholipid species, may lead to abnormal hunger-driven feeding.

    We agree that a western blot would be informative as well, but we were unable to obtain the reagent from Dr. Wang’s group, precluding us from performing this request. See email snapshot.

    To ensure that we appropriately discuss and clarify this issue, we have now included a section in the discussion - Page 14 Lines 26-34- under the subtitle “The implications of relationship between Pect levels and HSD”. We have pasted an excerpt from that subsection below for this reviewers assessment.

    Also, we note that over-expression of Pect cDNA in the fat-body does not alter phospholipid balance (Figure S9) and indeed improves HDF on HSD (Figure 7B). While this may appear inconsistent, it is critical to note that over-expression of Pect cDNA using UAS/Gal4 only increases Pect mRNA expression by 7-fold (Figure S6A), whereas HSD causes its upregulation by 250-fold (Figure S6B). Hence, we speculate that an increased ‘basal’ level of Pect such as by that provided by a cDNA over-expression in fat, may be protective to the negative effects of HSD (Figure 7B) without affecting overall phospholipid levels (Figure S9) , but extreme upregulation Pect on HSD affects the PE and LPE balance (Figure 3).”

    Reviewer #1 (Significance (Required)):

    The work is potentially novel and interesting, but at this stage it's difficult to interpret what the phenotype signifies. Although the manuscript could be revised simply by modifying the text, experimentally addressing the concerns would significantly improve the work.

    In sum, we hope we have addressed the key concern for Reviewer #1 as to whether the behavior we report here is indeed a dampening of starvation-induced feeding, or an effect of increase in baseline feeding. We hope that by reviewing our non-normalized data, they can appreciate that it is the former. Also, we hope that Reviewer #1 appreciates that we have strived to address the concerns by additional experiments, to clarify our findings and improve the impact of the work.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    This intriguing manuscript by Kelly and colleagues uses the fruit fly Drosophila melanogaster as a model to understand how diet-induced obesity alters the feeding response over time. In particular, the authors findings indicate that chronic exposure to a high-sugar diet significantly alters the starvation-induced feeding response. These behavioral studies are complemented by a lipidomics approach that reveals how a chronic high sugar affects many lipid species, including phospholipids. The authors then pursue mechanistic studies that indicate phospholipid metabolism within the fat body appears to remotely affect insulin secretion from the insulin producing cells. Moreover, the changes in phospholipid abundance are associated with changes in insulin-signaling, including increased insulin secretion from the IPCs and elevated levels of FOXO within the nucleus.

    I find the study to be potentially very important - the authors combine a longitudinal study that would be difficult in any other model with the powerful genetic tools available in the fly. The conclusions are mostly convincing, but a few follow-up experiments are required:

    We are grateful for the reviewers constructive, detail-oriented, and balanced feedback, and their recognition of the value of this study. Now, we have performed additional experiments to address the key concerns raised by all reviewers. We hope that on reading the revised version of our study, that the reviewer continues to feel positive about the message of this study and its potential impact.

    1. The key conclusions from the manuscript assume that manipulation of Pect expression levels alters phosphatidylethanolamine (PE) levels. However, the authors make no attempt to verify that the genetic experiments described herein actually affect PE levels. At a minimum, changes in PE levels should be verified for the Pect knockdown and overexpression lines. Similarly, there is no evidence that manipulation of either EAS or Pcyt2 induces the expected metabolic effects. I'm not asking that the longitudinal feeding experiments be repeated, simply that the authors measure the relevant lipid species, preferably with a targeted LC-MS approach.

    Prompted by this reviewer, we performed targeted LC-MS on whole adult flies, on normal diet, to assess lipid levels for fat-specific Pect-KD and overexpression. We decided to focus on Pect, as its knock-down even on normal diet causes a dampened hunger-driven feeding behavior (Figure 7A) and phenocopied a 14-day HSD feeding phenotype.

    We now present a new dataset in Figure 6. Two striking changes occur:

    They are:

    Pect-KD shows a significant reduction in specific class of PE 36.2 (Figure 6B). Our data regarding decrease in PE 36.2 agree with a previous lipidomic analyses of Pect mutant retina (PMID: 30737130). It is to be noted that though overall levels of all PE species trend downwards, like the Clandinin lab study on Pect (PMID: 30737130), we did not find a significant change in the overall PC and PE levels.

    Pect-KD shows increase in the phospholipid classes LPC and LPE (Figure 6A). In contrast, LPE is significantly downregulated on HSD Day 14 (Figure 3). On 14-day HSD consistent with extreme upregulation of Pect mRNA fed flies (Figure S6A; Pect mRNA 200-250 fold), PE trends upwards on 14-day HSD (Figure 3) and PE 36.2 trends higher (Figure S7C). We note that on the surface of it PE and LPE per se are contrasting between 14-day HSD lipidome and fat-specifc Pect-KD. But there is a significant commonality that under both states there is an imbalance of phospholipids classes PE and LPE. Hence, we propose that maintaining the compositional balance of phospholipid classes PE and LPE is critical to hunger-driven feeding and insulin sensitivity. Hence, either increase or decrease, of these key phospholipid species, may lead to abnormal hunger-driven feeding.

    Finally, fat-specific Pect-OE did not cause significant changes to lipid species (Figure S9). This could either be due to the fact that in fat-specific Pect-OE flies under normal food and that we were assaying whole body lipid levels and not fat-specific lipid changes. But to counter that, even a 60% reduction in Pect mRNA levels (Figure S6A), was sufficient to produce an effect on whole body phospholipid balance (Figure 6). Hence, we speculate that by maintaining a basally higher (7-fold higher Pect mRNA level Figure S6A), might allow 14-day HSD-fed flies to buffer the negative effects of HSD and we predict that it might take longer to disrupt the phospholipid balance and HDF response.

    We have now included a section in the discussion - Page 14 Lines 26-34- under the subtitle “The implications of relationship between Pect levels and HSD”. We have pasted an excerpt from that subsection below for this reviewers assessment.

    Also, we note that over-expression of Pect cDNA in the fat-body does not alter phospholipid balance (Figure S9) and indeed improves HDF on HSD (Figure 7B). While this may appear inconsistent, it is critical to note that over-expression of Pect cDNA using UAS/Gal4 only increases Pect mRNA expression by 7-fold (Figure S6A), whereas HSD causes its upregulation by 250-fold (Figure S6B). Hence, we speculate that an increased ‘basal’ level of Pect such as by that provided by a cDNA over-expression in fat, may be protective to the negative effects of HSD (Figure 7B) without affecting overall phospholipid levels (Figure S9), but extreme upregulation Pect on HSD affects the PE and LPE balance (Figure 3).”

    A central hypothesis in the study is that the HSD over a period of 14 days results in insulin resistant and that these changes are leading to changes in hunger dependent feeding. I would encourage the authors to determine if Foxo mutants are resistant to these HSD-induced effects on HFD.

    We thank the reviewers for this suggestion. However, given that dFOXO nuclear localization rather than expression levels regulate insulin sensitivity, we feel that disrupting dFOXO levels via mutation or knockdown will produce a plethora of indirect effects including developmental abnormalities (PMID: 24778227, PMID: 16179433, PMID: 29180716, PMID: 12893776). Our data suggest that chronic HSD treatment and Pect affect insulin sensitivity in fat tissue. However, we feel that investigating whether insulin sensitivity/FOXO signaling in fat tissue regulates feeding behavior is outside the scope of our work.

    1. In lines 25-30, the authors draw the conclusion that an increase in unsaturated fatty acid species is associated with the HSD and that these changes results in a more fluid lipid environment. While I agree with the model, the manuscript contains no evidence to support such a model. Either test the hypothesis or move the last line of the section to the discussion.

    We thank the reviewer for this important and insightful comment. We agree that the data we presented and discussed in the original version is at the moment speculative. Addressing the hypothesis that increase in unsaturated fatty acid species result in a more fluid lipid environment will require us to build tools and expertise. Hence, this hypothesis is better suited for exploration in a future study. Given this, we have moved this out of the results section into the Discussion section titled “HSD and fat-specific PECT-KD causes changes to phospholipid profile” (See excerpt below from page 13, lines 24-35).

    In addition to changes in phospholipid classes, we found that HSD caused an increase in the concentration of PE and PC species with double bonds (Figure S4C and S4D). Double bonds create kinks in the lipid bilayer, leading to increased lipid membrane fluidity which impacts vesicle budding, endocytosis, and molecular transport14,92. Hence it is possible that a mechanism by which HSD induces changes to signaling is by altering the membrane biophysical properties, such as by increased fluidity, which would have a significant impact on numerous biological processes including synaptic firing and inter-organ vesicle transport.”

    Also, as per the reviewer’s guidance, given that we are speculating here, we have also shifted this dataset from Main figure 4 to supplement S4C and S4D.

    In addition, lines 25-30 state that FFAs are increased after 14 days of a HSD. Figure 3A shows the exact opposite - FFAs are significantly decreased in 14 day fed animals despite being elevated in the 7 day fed animals. This is an interesting result that warrants discussion. Moreover, I would encourage to examine the lipidomic data more carefully to ensure that the text accurately portrays the lipid profiles.

    We apologize for misstating that FFAs are decreased on 14-day HSD in the lines 25-30. It was an error and we have corrected this. We agree with the reviewer that the reduction of FFA on Day 14-HSD is an intriguing and unexpected observation that needs to be emphasized and further discussed. To this end, we have added figure S4B, wherein we have provided the difference in FFA concentration (by species) after days 7 and 14.

    Furthermore, we have discussed what the potential meaning of reduced FFA at Day 14 implies in page 12, lines 19-27 of the Discussion section titled “HSD and fat-specific PECT-KD causes changes to phospholipid profile”. We have stated the following-

    We speculate that this reduction in FFA maybe due to their involvement in TAG biogenesis (PMID: 13843753). We were interested to see if the decrease in FFA correlated to a particular lipid species, as PE and PC are made from DAGs with specific fatty acid chains. However, further analysis of FFAs at the species level did not reveal any distinct patterns. The majority of FFA chains decreased in HSD, including 12.0, 16.0, 16.1, 18.0, 18.1, and 18.2 (Figure S4B). This data was more suggestive of a global decrease in FFA, likely being converted to TAG and DAG, rather than a specific fatty acid chain being depleted.”

    The processed lipidomics data should also be included as supplementary data table so that they can be independently analyzed by the reader.

    We thank the reviewer for this suggestion. As per the reviewers request, we have included the raw data as an attachment in our supplementary material (Supplementary Files 1-3.), so that interested readers can use the datasets generated in this study for future work and further analysis.

    Beyond these experimental suggestions, the manuscript needs significant editing for clarity. While I won't provide a comprehensive list, the authors need to provide accurate descriptions and annotation of genotypes (including w[1118], which is written as W1118), typos, and formatting. I've listed a few examples below:

    1. Page 3, Line 1 and 2: "...have been shown to impact feeding behavior and metabolism that leads to..." This is an awkward and grammatically incorrect sentence.
    2. Page 3, Lines 7-32 is one very large paragraph but contains concepts that should be broken down over at least three paragraphs.
    3. Page 3, Line 25: A description of the reaction catalyzed by Pect would be helpful for a manuscript focused on Pecte activity.
    4. Page 4, Line 10: "previously characterized method of eliciting diet induced feeding behavior." As stated in the text, the method is previously described yet the manuscript characterizing the method isn't cited.
    5. Figure legend 3 contains a random assortment of capitalized lipid species. Also, the names of lipid species are inappropriately broken into multiple names. Please use correct nomenclature throughout the manuscript.

    The list above is nowhere near comprehensive. The manuscript requires significant editing.

    We are grateful to the reviewer for drawing our attention to these errors. We have made significant edits to the revised manuscript to address the above-mentioned concerns, as well as made additional textual changes throughout and copyedited it. We hope that the reviewer will find the manuscript reads better and the clarity and preciseness is significantly improved.

    Reviewer #2 (Significance (Required)):

    I find the study to be potentially very important - the authors combine a longitudinal study that would be difficult in any other model with the powerful genetic tools available in the fly. The findings will significantly advance our understanding of how lipid metabolism links dietary nutrition with feeding behavior.

    Once again, we are grateful for this reviewer’s thoughtful critique and encouraging words regarding our work and its potential impact.

    __Reviewer #3 (Evidence, reproducibility and clarity (Required)): __

    Summary: This manuscript uses Drosophila to investigate how diet-induced obesity and the changes in the lipid metabolism of the fat boy modulate hunger-driven feeding (HDF) response. The authors first demonstrate that chronic exposure (14 days) of high sugar diet (HSD) suppresses HDF response. Through lipidome analysis, the authors identify a specific class of lipids to be elevated upon chronic HSD feeding. This coincided with the changes in expression of Pect, an enzyme that regulates the biosynthesis of these lipids. Modulating the expression of Pect specifically in the fat body affected HDF response.

    We thank this reviewer for their rigorous and thoughtful critique and for identifying a key issue with our original study pertaining to a gap in how Pect mRNA levels on 14-day HSD are elevated but the Pect-KD phenocopies the HDF. Now by performing whole-body adult fly lipidomic on fat-specific Pect-KD we have resolved this issue and provided clarity on role of Pect in maintaining phospholipid homeostasis and thus subsequently impacts hunger-driven feeding. We hope the reviewer finds that the revised manuscript provides further clarity to the functional link between Pect’s role in fat-body and hunger-driven feeding.

    Major comments: The author claim that the HDF response in HSD is distinct between early (5d, 7d) and chronic (day 14) HSD feeding. However, the data seem to indicate that HDF response is significantly decreased at all time points in HSD. For example, at day 5 HDF response was increased only 3-fold in HSD (Figure 1C) compared to around 50-fold increase in NF (Figure 1B). The scale of the Y-axis in Figure 1B and 1C is an order of magnitude different. Including the starved data (NFstv and HSDstv) in Figure S1, normalized to NF fed group, would better visualize the overall trends. Related to this, having the source data for the actual number of feeding events would be useful (e.g., to see the baseline changes in feeding in different time points in Figure 1 and the effect of genetic manipulations in Figure 7).

    As per the reviewers request, we now have modified our graphs to show source data (Figure S1) and show the raw feeding events.

    Then in the non-normalized graphs we plot, over a longitudinal time course, baseline and hunger-driven feeding events (Figure 1B-D). We also show that HSD fed flies do not display increased baseline feeding (Figure 1D) suggesting that the effect we see on HDF are no clouded by increased baseline feeding.

    Yes, the reviewer makes an important point that HDF response on HSD fed flies is of a lower magnitude than NF fed flies. We think that is a biologically meaningful observation, as it suggests that flies have a remarkably fine-tuned ability to coordinate food-intake with nutrient store levels.

    ­­Now we have included a paragraph in the Discussion, Page 11 Lines 23-27, that say the following to ensure the readers appreciate this salient point raised by this reviewer.

    *It is to be noted that the HDF response of HSD-fed flies (Figure 1C, Days 3-10) is of lower order of magnitude than the NF-fed flies. This suggests that that in addition to sensing an energy deficit and mobilizing fat stores (Figure 1F, 1G, S1), HSD fed flies calibrate their starvation-induced feeding to compensate only for the lost amount of fat. Overall, this suggests that flies have a remarkably fine-tuned ability to coordinate food-intake with nutrient store levels. *

    The association between fat body Pect level and phospholipid levels is not clear. Day 14 of HSD feeding shows high expression of Pect in the fat body and elevated levels of PC32.0 and PC32.2. The authors assume the high expression of Pect in the fat body is due to the compensatory response, but there are no data indicating downregulation of Pect levels at the earlier time points of HSD feeding. A previous study demonstrated that Pect mutant flies have lower levels of PC32.0 but higher PC32.2 (PMID: 30737130).

    We agree that one puzzling aspect of the original version of this study was that Pect mRNA levels being very high on Day 14 HSD, but nonetheless the effects of Pect-KD phenocopied HSD. To resolve this, prompted by Reviewer #2 and #3 concerns, for this revised version we have now performed lipidomic analyses on whole adult flies, when Pect is knocked down (KD) by RNAi in the fat tissue. We now present a new dataset in Figure 6. Two striking changes occu. They are:

    1. Pect-KD shows increase in the phospholipid classes LPC and LPE (Figure 6A). In contrast, LPE is significantly downregulated on HSD Day 14 (Figure 3).
    2. Pect-KD shows a significant reduction in specific class of PE 36.2 (Figure 6B). Our data regarding increase in PE 36.2 agree with a previous lipidomic analyses of Pect mutant retina (PMID: 30737130). In contrast, PE 36.2 trends upwards on 14 day HSD (Figure S7C) though not significantly. On 14-day HSD consistent with extreme upregulation of Pect mRNA fed flies (Figure S6A; Pect mRNA 200-250 fold), PE trends upwards on 14-day HSD (Figure 3) and PE 36.2 trends higher (Figure S7C). We note that on the surface of it PE and LPE per se are contrasting between 14-day HSD lipidome and fat-specifc Pect-KD. But there is a significant commonality that under both states there is an imbalance of phospholipids classes PE and LPE. Hence, we propose that maintaining the compositional balance of phospholipid classes PE and LPE is critical to hunger-driven feeding and insulin sensitivity. Hence, either increase or decrease, of these key phospholipid species, may lead to abnormal hunger-driven feeding.

    On day 14, HDF response was increased 70-fold in w1118 flies in NF (Figure 1B; w1118), but only 2.5-fold in lpp>LucRNAi control flies in NF (Figure 7A). This suggests that lpp-gal4 driver lines have a significant effect on HDF response. Using a different fat-body specific Gal4 line would be necessary to validate conclusions.

    Regards reduced HDF magnitude, in our experience using UAS-Gal4 reduces HDF response magnitude consistently and cannot be compared to w1118 which is more robust. To account for background differences, we use Uas-Gal4 with control RNAi. It clearly shows differences in HDF response on starvation, but Pect and Pisd RNAi does not (Figure 7A). Hence, given that this experiment internally controls for any changes in HDF response for UAS-Gal4>RNAi, we conclude that HDF response in disrupted in Pect and PISD KD (Figure 7).

    We only presented the Lpp-driver in our study, as this driver is the only fat-specific driver that has no leaky expression in other tissues, and is specific to fat as apolpp promoter used to generate this Gal4 line is only expressed in fat tissue (Eaton and colleagues, PMID: 22844248). Other widely used fat-specific drivers, including the pumpless-Gal4 (ppl-Gal4) driver has leaky expression in gut or other tissues (See Table 2 of this detailed study by Dr. Drummond- Barbosa https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7642949/). If the reviewer is aware of a fat-specific Gal4 line, other than Lpp-Gal4, which has a highly specific expression in the fat tissue without leaky expression in other tissues, then we are happy to take onboard the reviewer’s suggestion and try that fat-specific Gal4 that they suggest.

    HSD feeding promotes Pect expression (Figure S3C) and global changes in phospholipid levels (Figure 3, 4). Therefore, shouldn't Pect overexpression (not Pect RNAi) in a normal diet mimic HSD feeding state and promote loss of HDF response? Conversely shouldn't knockdown of Pect in HSD rescue loss of HDF response?

    We agree that a puzzling aspect is that Pect mRNA levels are significantly elevated in HSD Day-14, but Pect-KD showed displays the inappropriate HDF response. As we have described in our response to this reviewer on Page 19, we believe that Pect-KD and HSD disrupt PE and LPE balance overall but in different ways. Whereas Pect-OE using cDNA expression in fat body does not cause a significant change to any lipid class (Figure S9), and our results suggest that basally higher level of PECT is likely to be protective on HSD with respect to HDF(Figure 7B).

    To ensure that we appropriately discuss and clarify this issue, we have now included a section in the discussion - Page 14 Lines 26-33- under the subtitle “The implications of relationship between Pect levels and HSD”. We have pasted an excerpt from that subsection below for this reviewers assessment.

    Also, we note that over-expression of Pect cDNA in the fat-body does not alter phospholipid balance (Figure S9) and indeed improves HDF on HSD (Figure 7B). While this may appear inconsistent, it is critical to note that over-expression of Pect cDNA using UAS/Gal4 only increases Pect mRNA expression by 7-fold (Figure S6A), whereas HSD causes its upregulation by 250-fold (Figure S6B). Hence, we speculate that an increased ‘basal’ level of Pect such as by that provided by a cDNA over-expression in fat, may be protective to the negative effects of HSD (Figure 7B) without affecting overall phospholipid levels (Figure S9) , but extreme upregulation Pect on HSD affects the PE and LPE balance (Figure 3).”

    We would have liked to test Pect protein expression on HSD, but since we were unable to access antibodies for Pect published in a prior study (PMID: 33064773) from Dr. Wang’s lab (see Page 10-11, of response to Reviewer #1). Hence, we were unable to test how the proteins levels of Pect correlate with the 250-fold increase mRNA expression.

    In conclusion, we hope the reviewer appreciates that our results regarding Pect function are consistent with the main conclusion that achieving the right phospholipid balance between PE and LPE, is critical for an organism to display an appropriate HDF response.

    Minor comments: All graphs should plot individual data points and showed as box and whisker plot as much as possible.

    Thanks for this suggestion, we have added individual data points to the vast majority of figures in the paper. We have made exceptions to graphs such as seen in figure 1 and FigureS4B-D where we find individual data points add an unnecessary layer of complexity. We hope these changes provide additional clarity and strength to the claims made in this manuscript.

    Data for day 14 missing in Figure S4A and S4B.

    We have provided Day 14 for the PC composition and PE composition, due to changes in Figures, they are now S7A and S7B.

    Reviewer #3 (Significance (Required)):

    The interactions between diet-induced obesity, peripheral tissue homeostasis and feeding behavior is an interesting topic that can be addressed using Drosophila. This manuscript demonstrates how fat body Pect levels affect HSD induced changes in hunger-driven feeding response. However, at this point, the functional association between fat body Pect level, global phospholipid level, and loss of hunger-driven feeding response in chronic HSD feeding is not clear.

    We hope the revised data, and discussion of the paper, provides well-substantiated functional association on the importance of maintaining phospholipid balance, driven by Pect enzyme, as a critical regulator of hunger-driven feeding behavior. As stated in the revised discussion, the key take home message of our manuscript is that on prolonged HSD exposure PC, PE and LPE levels are dysregulated, the loss of phospholipid homeostasis coincided with a loss of hunger-driven feeding. Following this lead on phospholipid imbalance, we then uncovered a critical requirement for the activity of the rate-limiting PE enzyme PECT within the fat tissue in controlling hunger-driven feeding.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    This manuscript uses Drosophila to investigate how diet-induced obesity and the changes in the lipid metabolism of the fat boy modulate hunger-driven feeding (HDF) response. The authors first demonstrate that chronic exposure (14 days) of high sugar diet (HSD) suppresses HDF response. Through lipidome analysis, the authors identify a specific class of lipids to be elevated upon chronic HSD feeding. This coincided with the changes in expression of PECT, an enzyme that regulates the biosynthesis of these lipids. Modulating the expression of PECT specifically in the fat body affected HDF response.

    Major comments:

    The author claim that the HDF response in HSD is distinct between early (5d, 7d) and chronic (day 14) HSD feeding. However, the data seem to indicate that HDF response is significantly decreased at all time points in HSD. For example, at day 5 HDF response was increased only 3-fold in HSD (Figure 1C) compared to around 50-fold increase in NF (Figure 1B). The scale of the Y-axis in Figure 1B and 1C is an order of magnitude different. Including the starved data (NFstv and HSDstv) in Figure S1, normalized to NF fed group, would better visualize the overall trends. Related to this, having the source data for the actual number of feeding events would be useful (e.g., to see the baseline changes in feeding in different time points in Figure 1 and the effect of genetic manipulations in Figure 7).

    The association between fat body PECT level and phospholipid levels is not clear. Day 14 of HSD feeding shows high expression of pect in the fat body and elevated levels of PC32.0 and PC32.2. The authors assume the high expression of pect in the fat body is due to the compensatory response, but there are no data indicating downregulation of pect levels at the earlier time points of HSD feeding. A previous study demonstrated that pect mutant flies have lower levels of PC32.0 but higher PC32.2 (PMID: 30737130). To better understand the link the authors should knockdown/OE PECT specifically in the fat body and assess changes in phospholipids.

    On day 14, HDF response was increased 70-fold in w1118 flies in NF (Figure 1B; w1118), but only 2.5-fold in lpp>LucRNAi control flies in NF (Figure 7A). This suggests that lpp-gal4 driver lines have a significant effect on HDF response. Using a different fat-body specific Gal4 line would be necessary to validate conclusions.

    HSD feeding promotes PECT expression (Figure S3C) and global changes in phospholipid levels (Figure 3, 4). Therefore, shouldn't PECT overexpression (not PECT RNAi) in a normal diet mimic HSD feeding state and promote loss of HDF response? Conversely shouldn't knockdown of PECT in HSD rescue loss of HDF response?

    Minor comments:

    All graphs should plot individual data points and showed as box and whisker plot as much as possible. Data for day 14 missing in Figure S4A and S4B.

    Significance

    The interactions between diet-induced obesity, peripheral tissue homeostasis and feeding behavior is an interesting topic that can be addressed using Drosophila. This manuscript demonstrates how fat body PECT levels affect HSD induced changes in hunger-driven feeding response. However, at this point, the functional association between fat body PETC level, global phospholipid level, and loss of hunger-driven feeding response in chronic HSD feeding is not clear.

    Referees cross-commenting

    I agree with all the reviwers points; potentially very interesting, but requires a significant amount of work.

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    Referee #2

    Evidence, reproducibility and clarity

    This intriguing manuscript by Kelly and colleagues uses the fruit fly Drosophila melanogaster as a model to understand how diet-induced obesity alters the feeding response over time. In particular, the authors findings indicate that chronic exposure to a high-sugar diet significantly alters the starvation-induced feeding response. These behavioral studies are complemented by a lipidomics approach that reveals how a chronic high sugar affects many lipid species, including phospholipids. The authors then pursue mechanistic studies that indicate phospholipid metabolism within the fat body appears to remotely affect insulin secretion from the insulin producing cells. Moreover, the changes in phospholipid abundance are associated with changes in insulin-signaling, including increased insulin secretion from the IPCs and elevated levels of FOXO within the nucleus.

    I find the study to be potentially very important - the authors combine a longitudinal study that would be difficult in any other model with the powerful genetic tools available in the fly. The conclusions are mostly convincing, but a few follow-up experiments are required:

    1. The key conclusions from the manuscript assume that manipulation of PECT expression levels alters phosphatidylethanolamine (PE) levels. However, the authors make no attempt to verify that the genetic experiments described herein actually affect PE levels. At a minimum, changes in PE levels should be verified for the PECT knockdown and overexpression lines. Similarly, there is no evidence that manipulation of either EAS or Pcyt2 induces the expected metabolic effects. I'm not asking that the longitudinal feeding experiments be repeated, simply that the authors measure the relevant lipid species, preferably with a targeted LC-MS approach.
    2. A central hypothesis in the study is that the HSD over a period of 14 days results in insulin resistant and that these changes are leading to changes in hunger dependent feeding. I would encourage the authors to determine if Foxo mutants are resistant to these HSD-induced effects on HFD.
    3. In lines 25-30, the authors draw the conclusion that an increase in unsaturated fatty acid species is associated with the HSD and that these changes results in a more fluid lipid environment. While I agree with the model, the manuscript contains no evidence to support such a model. Either test the hypothesis or move the last line of the section to the discussion.

    In addition, lines 25-30 state that FFAs are increased after 14 days of a HSD. Figure 3A shows the exact opposite - FFAs are significantly decreased in 14 day fed animals despite being elevated in the 7 day fed animals. This is an interesting result that warrants discussion. Moreover, I would encourage to examine the lipidomic data more carefully to ensure that the text accurately portrays the lipid profiles.

    The processed lipidomics data should also be included as supplementary data table so that they can be independently analyzed by the reader.

    Beyond these experimental suggestions, the manuscript needs significant editing for clarity. While I won't provide a comprehensive list, the authors need to provide accurate descriptions and annotation of genotypes (including w[1118], which is written as W1118), typos, and formatting. I've listed a few examples below:

    1. Page 3, Line 1 and 2: "...have been shown to impact feeding behavior and metabolism that leads to..." This is an awkward and grammatically incorrect sentence.
    2. Page 3, Lines 7-32 is one very large paragraph but contains concepts that should be broken down over at least three paragraphs.
    3. Page 3, Line 25: A description of the reaction catalyzed by PECT would be helpful for a manuscript focused on PECT activity.
    4. Page 4, Line 10: "previously characterized method of eliciting diet induced feeding behavior." As stated in the text, the method is previously described yet the manuscript characterizing the method isn't cited.
    5. Figure legend 3 contains a random assortment of capitalized lipid species. Also, the names of lipid species are inappropriately broken into multiple names. Please use correct nomenclature throughout the manuscript.

    The list above is nowhere near comprehensive. The manuscript requires significant editing.

    Significance

    I find the study to be potentially very important - the authors combine a longitudinal study that would be difficult in any other model with the powerful genetic tools available in the fly. The findings will significantly advance our understanding of how lipid metabolism links dietary nutrition with feeding behavior.

    Referees cross-commenting

    I agree. We all think the manuscript is potentially interesting and important, but requires further experimentation. I agree with all concerns raised by the other reviewers. A revision would likely represent a significant amount of work.

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    Referee #1

    Evidence, reproducibility and clarity

    In this manuscript, Kelly et al. show that the difference between the feeding behavior of fed and starved flies (hunger-driven feeding; HDF) is absent in animals fed a high-sugar diet (HSD) for two weeks or more. The disappearance of HDF with HSD coincides with changes in phospholipid profiles caused by HSD. Furthermore, RNAi-mediated downregulation of PECT in the fat body-a key enzyme in the PE biosynthesis pathway-phenocopies physiological effects of HSD. Moreover, downregulation or overexpression in the fat body abolishes or induces HDF, respectively, abolishes or induces HDF, respectively, independent of HSD treatment.

    Overall, the manuscript is well-written and the phenotypes are clear. However, I have major concerns regarding the authors' interpretation of the data and their conclusion. Most importantly, while it is clear that the authors' high-sugar dietary treatment affects feeding behavior and physiology, I am not convinced that the changes can be considered "hunger-driven"-which is central to the main point of the manuscript. Therefore, it is my recommendation that the authors substantially revise the manuscript by either showing additional/re-analyzed data that rule out alternative hypotheses, or rewriting the manuscript keeping alternative interpretations in mind.

    Major comments:

    1. The data do not sufficiently show that the long-term HSD regime disrupts "hunger-sensing." The manuscript should address alternative hypotheses by showing raw instead of normalized data, rewriting the manuscript with a new central conclusion, or running additional experiments that actually show a defect in hunger-driven response.
      • a. The main results that the authors rely on for the argument is that the ratio of feeding events that the starved and non-starved flies eat is different between the groups fed normal or HSD. However, because the authors only show normalized data (normalized to non-starved flies; Fig. 1), it is difficult to tell whether the change is due to a chronically increased feeding in non-starved HSD flies-maybe in perpetual hunger-like allostasis-or dampened starvation response. Indeed, the data shown in Fig S1 show that flies fed HSD for as short as 5 days show more frequent feeding events compared to age-matched controls fed normal food. It is possible that because the HSD-fed flies eat more than NF-fed flies, even without being starved, the ratio of starved/non-starved feeding is lower in the HSD-fed group-due to changes in the denominator, rather than the numerator.
      • b. It is also possible that the HSD-fed flies are simply not in as big an energy deficit physiologically, due to the increased fat deposits they've accumulated (as the authors show later in the manuscript). It may take longer for the fat HSD flies to reach substantial energy deficiency than the NF flies, but they still may eventually be able to appropriately respond to hunger, just like NF flies. In such case, it would be a misnomer to call this behavioral change a 'defect in hunger-driven feeding behavior.' Maybe an experiment with a dose-response curve of "hunger driven feeding response" as a function of duration of starvation would help?
    2. How can you be sure that lower Dilp5 immunofluorescence is indicative of increased Dilp5 secretion? Wouldn't decreased production of dilp5 also have the same results?
      • a. Also, the authors should state in the main text that it is Dilp5, not just any Dilp.
    3. Data presentation:
      • a. Sometimes the data are normalized to NF (Fig 4B-C), sometimes not (ex. Fig 4A, S4C). Unless there is a specific rationale for the data transformation, it would be more appropriate to show untransformed data (ex. Fig 4A, S4C), especially as the authors use two-way ANOVA to determine significance. Only showing the differences implies comparison against a hypothetical mean (i.e. μ0=0), not between two group means.
      • b. Some figures show both individual data points and summary statistics (mean, SD, ... ex. Fig 2A)-which I believe is ideal-but some show only one or the other (ex. Fig 2B, no summary statistics; Fig. 3, no data points. The manuscript would read more convincing if data visualization is consistent across figures.

    Minor comments:

    1. High sugar diet: what is the actual sugar concentration in the NF v. HSD diets? The authors write that the HSD diet contains "30% more sugar" than the NF, but providing the final sugar concentrations-sucrose or others-would be informative for other scientists studying the effect of high sugar diets.
      • a. Additionally, the definition of HSD is inconsistent. Main text (Page 5, line 17) states that their HSD is "60% more sugar than normal media," whereas the figure legend (Fig 1) and the Methods state that the HSD contains "30% more sugar."
    2. Starvation medium: please provide justification for why the authors used 1% sucrose/agar for starvation medium, instead of plain agar/water that most labs use. At least clarify and provide a reference for the claim that the 1% sucrose/agar "is a minimal food media to elicit a starvation response."
    3. PECT mRNA level is higher with HSD. This is surprising because not only, as authors mention, is increased PC32.2 with HSD suggests lower PECT activity, but also because PECT RNAi phenocopies long-term HSD in HDF behavior, lipid morphology, FOXO accumulation in fat body. The authors speculate that the data "likely shown an upregulation in an attempt to mediate the PECT dysregulation occurring at the protein level." If that were true, a western blot may be informative. Zhao and Wang (2020, PLoS Genetics) generated a PECT antibody that seems compatible with western blot applications. That being said, I don't think such data is critical for the manuscript. I mention this simply as a suggestion for the authors.
      • a. page 8, line 22-23, did you mean to write "Given how PC32.2 is elevated after 14 days of exposure to HSD, we assumed that PECT levels would be low for flies under HSD," not "high?" Otherwise the subsequent 2 sentences don't make sense.

    Significance

    The work is potentially novel and interesting, but at this stage it's difficult to interpret what the phenotype signifies. Although the manuscript could be revised simply by modifying the text, experimentally addressing the concerns would significantly improve the work.

    The co-reviewer and I have expertise in Drosophila neurobiology and behavior.

    Referees cross-commenting

    Hi all, although the reviews hit upon some overlapping, but mostly different points, I agree with all of the concerns raised. There's some really interesting stuff here but some of the results, as presented, don't make sense. It's possible this will be clarified by revising the text, although I suspect it's more likely that the authors will have to add a number of the experimental suggestions made by the reviewers.

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