Mitochondrial fusion and altered beta-oxidation drive muscle wasting in a Drosophila cachexia model

  1. Callum Dark
  2. Nashia Ali
  3. Sofia Golenkina
  4. Ronnie Blazev
  5. Benjamin L Parker
  6. Katherine Murphy
  7. Gordon Lynch
  8. Tarosi Senapati
  9. S Sean Millard
  10. Sarah M Judge
  11. Andrew R Judge
  12. Louise Y Cheng

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

Cancer cachexia is a tumour-induced wasting syndrome, characterised by extreme loss of skeletal muscle. Defective mitochondria can contribute to muscle wasting; however, the underlying mechanisms remain unclear. Using a Drosophila larval model of cancer cachexia, we observed enlarged and dysfunctional muscle mitochondria. Morphological changes were accompanied by upregulation of beta-oxidation proteins and depletion of muscle glycogen and lipid stores. Muscle lipid stores were also decreased in Colon-26 adenocarcinoma mouse muscle samples, and expression of the beta-oxidation gene CPT1A was negatively associated with muscle quality in cachectic patients. Mechanistically, mitochondrial defects result from reduced muscle insulin signalling, downstream of tumour-secreted insulin growth factor binding protein (IGFBP) homolog ImpL2. Strikingly, muscle-specific inhibition of Forkhead box O (FOXO), mitochondrial fusion, or beta-oxidation in tumour-bearing animals preserved muscle integrity. Finally, dietary supplementation with nicotinamide or lipids, improved muscle health in tumour-bearing animals. Overall, our work demonstrates that muscle FOXO, mitochondria dynamics/beta-oxidation and lipid utilisation are key regulators of muscle wasting in cancer cachexia.

Article activity feed

  1. Review Commons

    Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Reply to the reviewers

    We would like to thank the reviewers for their insightful comments. We believe that the changes that have been suggested will add greatly to this paper, and we will endeavor to incorporate as many of these suggestions as we can.

    Reviewer #1

    This is an interesting study, which presents yet another mechanism involved in the regulation of tumour associated paraneoplastic syndromes, such as muscle wasting. It suggest the intriguing possibility of using a hight fat diet and modulating mitochondrial metabolism as a means of alleviating cachectic muscle wasting. However, as it stands, these aspects of the study remains rather preliminary. This is particularly the case regarding the role of dietary interventions in the model and understanding of the type of metabolic reprogramming in wasting muscles, which lack direct experimental evidence. If the authors were able to further develop this aspects of the study with robust experimental work, it will make it a very valuable and impactful report.

    1- All the mitochondrial phenotypes presented should be compared in the two different tumour models (Gal4/UAS and the QF/QUAS driven), which are indistinctively used throughout the study.

    We will ensure that mitochondrial size and TMRE staining are performed in the two different tumour models so that they can be compared.

    2- The mitochondrial phenotype of wasting muscles is only evident towards the late stages of tumourigenesis (7 day old larvae). Mitochondria of 5 day old tumour bearing animals is indistinct from the control ones. Given that 5 days is the oldest wild type larvae available, the authors need to assess the mitochondrial size and function in muscles form developmentally delayed, no-tumour bearing larvae to discard a trivial contribution of failed metamorphosis in such phenotype.

    We will examine mitochondrial size and TMRE in pmhGal4 > torsoRNAi animals (which undergo delayed metamorphosis) compared with control animals.

    4- TMRE staining presented in Figure 1 is not convincing. If available, a biochemical and/or more quantitative method to address mitochondrial function should be used.

    We will perform ATP synthesis and O2 consumption assays to provide a biochemical method to accompany the TMRE assays.

    5- Related to the point above. The extent of the mitochondrial phenotype following genetic manipulations in the tumour or muscle is not consistently analysed. In some cases, mitochondrial size and activity is assessed but in multiple cases, only mitochondrial size is measured. Mitochondrial activity should be assessed in all cases also.

    We will assess mitochondrial activity in a time course of RasV12DlgRNAi vs w1118, as well as tumor-bearing animals treated with nicotinamide, QF-QUAS RasV12scribRNAi, MHC> foxoRNAi, and RasV12DlgRNAi > Impl2RNAi.

    6- Are mitochondrial fusion proteins such as Marf upregulated in muscles undergoing wasting in Rasv12dlg RNAi animals?

    Regulation of neither Opa1 nor Marf are altered in our proteomics study.

    7- Is overexpression of mitochondrial fusion proteins alone sufficient to induce muscle wasting?

    No, overexpression of Marf was not sufficient to induce muscle wasting, however overexpression of Marf caused worsened muscle wasting in tumour-bearing animals. We will include this data in our revised manuscript.

    8- Is there a change in the expression of ATP5A in the muscles of bearing animals RasV12dlgRNAi, which has dysfunctional mitochondria compared to the control?

    There is no change in ATP5A expression in our proteomics study.

    9- Regarding measures of insulin signaling activity in muscle (Figure 2): the data provide on FOXO staining is not very convincing. Improved staining and robust and more quantitative measure of insulin signaling activity, such as western blot analysis of pAkt should be provided. Apart from the nucleus, there is an overall increase in FOXO expression in the muscle cells of RasV12dlgRNAi compared to the control. In control animals, there is no signal of FOXO. How do you explain this?

    We have attempted western blots of pAkt in tumour-bearing muscle previously and found that tumour metastases caused unreliable results, making immuno-staining a more reliable option. However, pAkt antibody staining also does not work well in the muscles. The control image we displayed was an extreme example, so we will choose more representative images that show more consistent FOXO staining.

    12- In S3 J-L, Since MHC expression is also dependent upon muscle health and integrity, it would be better to use another, and more universal, readout for protein translation/synthesis. For example, labelling the tissue with Puromycin or staining for translation initiation factors.

    We will perform O-propargyl-puromycin (OPP) staining for a w1118 vs RasV12DlgRNAi time course to provide another translation readout to accompany the MHC staining.

    13- How does lipid/high fat diet restore muscle wasting? What happens to the tumours of high fat and Nicotinamide feed animals? In all cases, the impact on tumour size upon genetic manipulations of the muscle should be shown.

    We will measure tumour size in tumour-bearing animals on both nicotinamide and high-fat diets, as well as QF-QUAS RasV12scribRNAi MHC> foxoRNAi, marfRNAi and whdRNAi animals. Impl2RNAi in tumour-bearing animals has been shown already (Lodge et al., 2021).

    14- Does NAM feeding or High-fat diet restore whd transcript levels??

    We will perform qPCR to examine whd transcript levels in tumour-bearing animals on nicotinamide diets as well as high-fat diets.

    15- Do these feeding regimes restore insulin signaling in RasV12dlgRNAi animals?

    We have demonstrated that for RasV12dlgRNAi animals fed a nicotinamide diet, FOXO levels are decreased (Fig 5D). We will do the same experiment for tumour-bearing animals fed a high fat diet.

    17- Related to the point above, DAPI and phalloidin should be included when showing lipid staining to understand better the cellular structures present in the field of view along with the lipid droplets.

    DAPI and phalloidin staining is not compatible with lipid staining, as they require the use of PBST (detergent) which breaks down extracellular lipids. We will include more representative, raw images in which the details of the muscle can be seen.

    Minor comments

    1. The order of panels in the figures and the main text should be the same for better readability.

    We will revisit the figures to ensure readability is improved.

    1. Figure S3 G-H: The image looks out of focus. Is Atg8 expression high near to the nucleus?

    Atg8a expression is highest near the nucleus, and is decreased in RasV12dlgRNAi > Impl2RNAi animals. We will provide more representative images to make this clearer.

    Reviewer #2

    This manuscript proposes and interesting new mechanism how tumours non-autonomously induce muscle mass loss (cachexia) in a genetic Drosophila model. These effects can be modified by diet. Hence results are interesting for both basic and more clinically interested audience.
    The weak point of the paper is the limited quantification of mitochondria sizes/morphologies, which is an important point that asks for significant improvement of either the imaging conditions or the image analysis.

    1. The authors provide evidence that eye or imaginal disc tumours induce larger mitochondria in muscles. The authors try to quantify mitochondrial sizes using an automated analysis. This is a tricky task from their light microscopy images that appear to be limited in resolution. By looking at the Suppl. Figure 1, I wonder how relevant an increase of a "large" mitochondria fraction from 7 to 12 % is in the tumour larvae, considering that a significant fraction of the mitochondria are currently not counted, as they are too large to be investigated (white colours in S1F, G). Can the authors increase resolution to resolve these large clumps that likely consist of individual mitochondria to reliably segment all of them, and not only a sub fraction. It would be useful to display the size profiles of all mitochondria in various conditions and not only of a very selected subset of "large" mitochondria. This comment applies to all figures in which mitochondria size was quantified and hence is critical for the entire manuscript.

    We will utilise a newly developed segmentation and centroid tracking-based analysis pipeline based in MATLAB, that may be able to separate the large clumps of mitochondria, to ensure that as many mitochondria can be quantified as possible. We will also provide size profiles of all mitochondria sizes from all conditions in which we performed mitochondria size analysis.

    1. Comparing MitoTracker to TMRE is a valid approach to estimate mitochondria activity/health. The images shown in 1H,I are overview images that seem to show large regional differences in the muscles of unclear origin. High resolution images of representative regions as shown for the ATP5A stains would be more convincing as these can resolve individual mitochondria to hopefully see damaged ones next to normal ones. Would "active" mitochondria not be expected to be the ones that oxidise a lot of fatty acid break down products?

    We will take representative zoomed in images for 1H & I to better demonstrate mitochondria morphology.

    1. The authors find that co-overexpressing FOXO in muscles results in a more severe muscle degeneration phenotype in tumour bearing animals than tumour alone. However, it seems the important control of FOXO overexpression in an otherwise wildtype animal is missing. In order to judge if the muscles really detach in these genotypes, instead of shrink and finally rupture, high resolution images of muscle attachment sites would be needed.

    We will assess if MHCGal4 > UAS dFOXO causes loss of muscle integrity. In addition, in both wildtype and tumour-bearing animals, we will overexpress FOXO in the muscles and stain for muscle attachment proteins such as tiggrin to determine if the phenotype seen is caused by a mislocalisation of proteins at attachment sites.

    1. The strongly reduced lipid droplets in the tumour bearing animals is interesting. To better normalise for the reduced size of the muscles, a counter staining for muscle and a following normalisation would make the statement stronger and thus better support the conclusion.

    As mentioned above we will provide more representative images to help visualize muscle structures in LipidTOX experiments. In addition, we will normalize the amount of lipid droplets detected to a set area, as opposed to just measuring total lipid droplets.

    Reviewer #3

    The strength of the study is the use of suitable in vivo model systems, combined with genetic manipulations to study the mechanisms behind cancer cachexia. The weak points of the study is the lack of functional assays such as quantitative measurements of oxidative phosphorylation and metabolites.

    1, Throughout the manuscript the authors use TMRE staining to evaluate mitochondrial function. To me it is not clear what function they are actually referring to. I assume they mean respiration/respiratory chain function, as this generates the proton motif force measured, but neither oxygen consumption nor aerobic ATP synthesis is ever mentioned or measured. Especially considering that the authors suggest that an increased flux through beta oxidation, which is a mitochondrial function, results in muscle wasting, the authors might want to consider measuring respiration with different substrates, using either a seahorse or Oroboros or equivalent.

    We do not have the necessary equipment or resources to perform Seahorse or Oroboros experiments. Therefore, we will perform O2 consumption and ATP synthesis assays for RasV12dlgRNAi and QF-QUAS RasV12scribRNAi vs w1118, RasV12dlgRNAi > Impl2RNAi, QF-QUAS RasV12scribRNAi > marfRNAi, whdRNAi, and tumour-bearing animals fed high fat diets to provide more insights into mitochondria function.

    3, It is difficult to understand that it is even possible for beta oxidation to exceed the capacity of the OXPHOS system. In that case one would have excess of acetyl CoA and NADH, inevitably inhibiting further beta oxidation and the TCA cycle due to lack of NAD, as well as numerous regulatory mechanisms. Additionally, one would expect increased ketone body production. The authors might want to clarify how the excess redox potential, due to increased beta oxidation is utilised.

    We will perform acetyl-CoA and NAD/NADH assays in RasV12dlgRNAi and QF-QUAS RasV12scribRNAi vs w1118 to determine if beta-oxidation is occurring in excess. In addition, we will clarify in the text that we hypothesize that increased beta-oxidation is utilizing the muscle’s resources to the point that there is none left to continue energy production.

    Minor:

    Line 223 "Together, this data suggests that FOXO lies upstream of beta-oxidation, and mitochondria function lies downstream of beta-oxidation".
    I would suggest to rephrase. Of course beta-oxidation and the TCA takes place inside mitochondria, so what mitochondrial functions do the authors refer to?

    As mentioned earlier, we will perform O2 consumption and ATP synthesis assays to strengthen this claim. In addition, we will rephrase this sentence to avoid confusion.

    Line 238 "Overall, this data suggests that the depletion of muscle lipid stores via beta oxidation affects mitochondrial function and is negatively correlated with muscle health in cachectic flies, mice and patients" - The mechanism is not fully clear to me as other energy sources are still available to the fly. The authors might want to expand here.

    We will clarify that there may be other energy sources available that were not investigated in this paper.

    Line 93 : "To test whether this increase in mitochondrial size could lead to compromised mitochondrial function, we performed live staining with tetramethylrhodamine ethyl ester (TMRE), a compound used to measure the membrane potential of mitochondria." - I am not sure that size on its own correlates with mitochondrial function, but rather the energetic and metabolic state of the cell. Increased biogenesis is a common response to dysfunction, but often reflected in increased mass.

    We will clarify the that the increase in size may be a reflection of increased metabolic need of the muscle.

    1. Description of the revisions that have already been incorporated in the transferred manuscript

    Please insert a point-by-point reply describing the revisions that were already carried out and included in the transferred manuscript. If no revisions have been carried out yet, please leave this section empty.

    Reviewer 1:

    3- In all cases, the age of experimental animals must be clearly indicated in figures and/or figure legends.

    We have already put the ages of the experimental animals in the bottom of the figure legends.

    11- Does insulin signaling influence Lipid metabolism in muscle?

    We demonstrate in the manuscript that FoxoRNAi in the muscle of tumour-bearing animals reduces whd transcript levels (Fig 4C), and Impl2RNAi in the tumour restores muscle lipid droplet levels (Fig 3G-I).

    2. Description of analyses that authors prefer not to carry out

    Please include a point-by-point response explaining why some of the requested data or additional analyses might not be necessary or cannot be provided within the scope of a revision. This can be due to time or resource limitations or in case of disagreement about the necessity of such additional data given the scope of the study. Please leave empty if not applicable.

    Reviewer 1:

    10- The phenotype of increased fatty acid oxidation in wasting muscles is inferred as per the proteomic signature but not directly demonstrated. TCA metabolite tracing using 13C-Palmitate should be used to demonstrate this, which is a central point of the manuscript.

    The examination of 13C-palmitate would require metabolomic approaches, for which we do not have the necessary equipment and is beyond our timeframe. Thus, we will aim to examine changes in mitochondria metabolism through other measures mentioned above.

    16- The lipid phenotype in cachectic fly muscles is not consistent with that reported in humans and shown by the authors in their xenograft model. While loss of lipid droplets is observed in the fly muscle cells, there is increase in the lipid content within the mouse muscle and only extramyocellular lipid is decreased. The relevance of the extracellular lipid is unclear.

    We hypothesize that this is due to a transport of lipids from extracellular lipid droplets to mitochondria for utilization, as has been suggested previously (Rambold et al., 2015). Examining in detail if this is the case in our models is beyond the scope of this paper.

    Reviewer 3:

    2, The authors suggest that an increase in beta oxidation exceeds mitochondrial function (?), which in turn induces a change in mitochondrial morphology, further contributing to the muscle wasting. The authors may want to demonstrate that there is indeed excess beta oxidation, by measuring a toxic accumulation of different lengths of acylcarnitines. For instance, it is well known that patients with beta oxidation defects accumulate toxic intermediates of beta oxidation that can ultimately affect mitochondrial function.
    The manuscript would be much improved if oxygen consumption is measured and combined with analysis of acylcarnitines.

    The examination of acylcarnitines would require lipidomic approaches, and is beyond our timeframe for these revisions. To try to address the need for investigations if beta-oxidation is in excess, we will perform oxygen consumption assays as mentioned and alter the manuscript to de-emphasize excess beta-oxidation.

    4, Unfortunately the supplementary information is in a format I can't open, thus I can't evaluate the method for identifying large mitochondria and other results in these files. This makes part of the reviewing process difficult.

    N/A

  2. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #3

    Evidence, reproducibility and clarity

    In this manuscript the authors study the mechanisms behind cancer cachexia, using drosophila cancer models. They find that muscle wasting in cachexia is mediated via two different mechanisms: either via insulin signalling and FOXO activation or beta oxidation via mitochondrial fusion.
    It is well known that many cancers can induce a catabolic state, compatible with a decrease in insulin signalling and one of the mechanisms proposed. Additionally, the authors suggest that an imbalance between mitochondrial capacity and beta oxidation flux leads to muscle wasting.

    Major comments:

    1. Throughout the manuscript the authors use TMRE staining to evaluate mitochondrial function. To me it is not clear what function they are actually referring to. I assume they mean respiration/respiratory chain function, as this generates the proton motif force measured, but neither oxygen consumption nor aerobic ATP synthesis is ever mentioned or measured. Especially considering that the authors suggest that an increased flux through beta oxidation, which is a mitochondrial function, results in muscle wasting, the authors might want to consider measuring respiration with different substrates, using either a seahorse or Oroboros or equivalent.
    2. The authors suggest that an increase in beta oxidation exceeds mitochondrial function (?), which in turn induces a change in mitochondrial morphology, further contributing to the muscle wasting. The authors may want to demonstrate that there is indeed excess beta oxidation, by measuring a toxic accumulation of different lengths of acylcarnitines. For instance, it is well known that patients with beta oxidation defects accumulate toxic intermediates of beta oxidation that can ultimately affect mitochondrial function.
      The manuscript would be much improved if oxygen consumption is measured and combined with analysis of acylcarnitines.
    3. It is difficult to understand that it is even possible for beta oxidation to exceed the capacity of the OXPHOS system. In that case one would have excess of acetyl CoA and NADH, inevitably inhibiting further beta oxidation and the TCA cycle due to lack of NAD, as well as numerous regulatory mechanisms. Additionally, one would expect increased ketone body production. The authors might want to clarify how the excess redox potential, due to increased beta oxidation is utilised.
    4. Unfortunately the supplementary information is in a format I can't open, thus I can't evaluate the method for identifying large mitochondria and other results in these files. This makes part of the reviewing process difficult.

    Minor:

    Line 223 "Together, this data suggests that FOXO lies upstream of beta-oxidation, and mitochondria function lies downstream of beta-oxidation".
    I would suggest to rephrase. Of course beta-oxidation and the TCA takes place inside mitochondria, so what mitochondrial functions do the authors refer to?

    Line 238 "Overall, this data suggests that the depletion of muscle lipid stores via beta oxidation affects mitochondrial function and is negatively correlated with muscle health in cachectic flies, mice and patients" - The mechanism is not fully clear to me as other energy sources are still available to the fly. The authors might want to expand here.

    Line 93 : "To test whether this increase in mitochondrial size could lead to compromised mitochondrial function, we performed live staining with tetramethylrhodamine ethyl ester (TMRE), a compound used to measure the membrane potential of mitochondria." - I am not sure that size on its own correlates with mitochondrial function, but rather the energetic and metabolic state of the cell. Increased biogenesis is a common response to dysfunction, but often reflected in increased mass.

    Significance

    General assessment: The strength of the study is the use of suitable in vivo modelsystems, combined with genetic manipulations to study the mechanisms behind cancer cachexia. The weak points of the study is the lack of functional assays such as quantitative measurements of oxidative phosphorylation and metabolites.

    Advance: The main advance of this study is attributed to mechanistic insights behind cancer cachexia and the role of mitochondria in more conditions as opposed to the its involvement in inherited mitochondria disease.

    Audience: This report should be of interest to a broad audience since it's studying a condition connected to cancer and cancer metabolism.

    Reviewers field of expertise: Mitochondrial disease/dysfunction, in vivo modelling, molecular biology, bioenergetics and metabolism

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    Chen and colleagues are using the Drosophila larval muscles model to investigate how a tumour can non-autonomously induce muscle mass loss, a known phenomenon called cancer cachexia. They report that tumours change muscle mitochondria morphologies, specifically their size and their chemistry. These changes correlate with increase in fat metabolism and a depletion of fat and glycogen reserves. Regarding the molecular mechanism, the authors propose that tumour cells secrete IGF binding protein that reduces the level of insulin and thus insulin signalling in muscle. They test this hypothesis by reducing FOXO activity, a negative regulator of insulin signalling, or mitochondrial fusion in muscles of tumour carrying larvae, which indeed appears to result in muscle improvements. These insights from Drosophila muscles suggest that tumour-caused reduced insulin signalling in muscles can be responsible for tumour induced muscle loss. A similar mechanism may apply to mammals and hence these findings are of clinical interest.

    Major comments

    1. The authors provide evidence that eye or imaginal disc tumours induce larger mitochondria in muscles. The authors try to quantify mitochondrial sizes using an automated analysis. This is a tricky task from their light microscopy images that appear to be limited in resolution. By looking at the Suppl. Figure 1, I wonder how relevant an increase of a "large" mitochondria fraction from 7 to 12 % is in the tumour larvae, considering that a significant fraction of the mitochondria are currently not counted, as they are too large to be investigated (white colours in S1F, G). Can the authors increase resolution to resolve these large clumps that likely consist of individual mitochondria to reliably segment all of them, and not only a sub fraction. It would be useful to display the size profiles of all mitochondria in various conditions and not only of a very selected subset of "large" mitochondria.
      This comment applies to all figures in which mitochondria size was quantified and hence is critical for the entire manuscript.
    2. Comparing MitoTracker to TMRE is a valid approach to estimate mitochondria activity/health. The images shown in 1H,I are overview images that seem to show large regional differences in the muscles of unclear origin. High resolution images of representative regions as shown for the ATP5A stains would be more convincing as these can resolve individual mitochondria to hopefully see damaged ones next to normal ones. Would "active" mitochondria not be expected to be the ones that oxidise a lot of fatty acid break down products?
    3. The authors find that co-overexpressing FOXO in muscles results in a more severe muscle degeneration phenotype in tumour bearing animals than tumour alone. However, it seems the important control of FOXO overexpression in an otherwise wildtype animal is missing. In order to judge if the muscles really detach in these genotypes, instead of shrink and finally rupture, high resolution images of muscle attachment sites would be needed.
    4. The strongly reduced lipid droplets in the tumour bearing animals is interesting. To better normalise for the reduced size of the muscles, a counter staining for muscle and a following normalisation would make the statement stronger and thus better support the conclusion.

    Significance

    This manuscript proposes and interesting new mechanism how tumours non-autonomously induce muscle mass loss (cachexia) in a genetic Drosophila model. These effects can be modified by diet. Hence results are interesting for both basic and more clinically interested audience.
    The weak point of the paper is the limited quantification of mitochondria sizes/morphologies, which is an important point that asks for significant improvement of either the imaging conditions or the image analysis.

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    Summary

    Larvae bearing RasV12; dlgRNAi eye tumours recapitulate aspects of cachexia, such as muscle wasting. In this manuscript, the authors use their previously characterized RasV12; dlgRNAi larval model of cancer cachexia to show that tumour induced cachectic muscle wasting is associated with excessive mitochondrial fusion, resulting in the formation of enlarged dysfunctional mitochondria in wasted muscle cells. Muscle specific blockade of mitochondrial fusion prevents muscle wasting and restores mitochondrial potential in tumour bearing animals. The authors also link increased mitochondrial size to decreased insulin signaling (increased foxo) caused by the tumour induced pro-cachexia factor and insulin inhibitor Impl2. Consistently, downregulation of ImpL2 from the tumour decreases foxo levels in muscle and reduces mitochondrial size. Finally, the authors show that wasting muscles in flies show decrease lipid droplets and a molecular and proteomic signature indicative of increased fatty acid oxidation. Muscle wasting, loss of lipids and mitochondrial integrity can be restored upon inhibition of Impl2 in the tumour, downregulation of the mitochondrial lipid transporter CPT1 or feeding animals with a high fat diet.

    Major comments

    1. All the mitochondrial phenotypes presented should be compared in the two different tumour models (Gal4/UAS and the QF/QUAS driven), which are indistinctively used throughout the study.
    2. The mitochondrial phenotype of wasting muscles is only evident towards the late stages of tumourigenesis (7 day old larvae). Mitochondria of 5 day old tumour bearing animals is indistinct from the control ones. Given that 5 days is the oldest wild type larvae available, the authors need to assess the mitochondrial size and function in muscles form developmentally delayed, no-tumour bearing larvae to discard a trivial contribution of failed metamorphosis in such phenotype.
    3. In all cases, the age of experimental animals must be clearly indicated in figures and/or figure legends.
    4. TMRE staining presented in Figure 1 is not convincing. If available, a biochemical and/or more quantitative method to address mitochondrial function should be used.
    5. Related to the point above. The extent of the mitochondrial phenotype following genetic manipulations in the tumour or muscle is not consistently analysed. In some cases, mitochondrial size and activity is assessed but in multiple cases, only mitochondrial size is measured. Mitochondrial activity should be assessed in all cases also.
    6. Are mitochondrial fusion proteins such as Marf upregulated in muscles undergoing wasting in Rasv12dlg RNAi animals?
    7. Is overexpression of mitochondrial fusion proteins alone sufficient to induce muscle wasting?
    8. Is there a change in the expression of ATP5A in the muscles of bearing animals RasV12dlgRNAi, which has dysfunctional mitochondria compared to the control?
    9. Regarding measures of insulin signaling activity in muscle (Figure 2): the data provide on FOXO staining is not very convincing. Improved staining and robust and more quantitative measure of insulin signaling activity, such as western blot analysis of pAkt should be provided. Apart from the nucleus, there is an overall increase in FOXO expression in the muscle cells of RasV12dlgRNAi compared to the control. In control animals, there is no signal of FOXO. How do you explain this?
    10. The phenotype of increased fatty acid oxidation in wasting muscles is inferred as per the proteomic signature but not directly demonstrated. TCA metabolite tracing using 13C-Palmitate should be used to demonstrate this, which is a central point of the manuscript.
    11. Does insulin signaling influence Lipid metabolism in muscle?
    12. In S3 J-L, Since MHC expression is also dependent upon muscle health and integrity, it would be better to use another, and more universal, readout for protein translation/synthesis. For example, labelling the tissue with Puromycin or staining for translation initiation factors.
    13. How does lipid/high fat diet restore muscle wasting? What happens to the tumours of high fat and Nicotinamide feed animals? In all cases, the impact on tumour size upon genetic manipulations of the muscle should be shown.
    14. Does NAM feeding or High-fat diet restore whd transcript levels??
    15. Do these feeding regimes restore insulin signaling in RasV12dlgRNAi animals?
    16. The lipid phenotype in cachectic fly muscles is not consistent with that reported in humans and shown by the authors in their xenograft model. While loss of lipid droplets is observed in the fly muscle cells, there is increase in the lipid content within the mouse muscle and only extramyocellular lipid is decreased. The relevance of the extracellular lipid is unclear.
    17. Related to the point above, DAPI and phalloidin should be included when showing lipid staining to understand better the cellular structures present in the field of view along with the lipid droplets.

    Minor comments

    1. The order of panels in the figures and the main text should be the same for better readability.
    2. Figure S3 G-H: The image looks out of focus. Is Atg8 expression high near to the nucleus?

    Significance

    This is an interesting study, which presents yet another mechanism involved in the regulation of tumour associated paraneoplastic syndromes, such as muscle wasting. It suggest the intriguing possibility of using a hight fat diet and modulating mitochondrial metabolism as a means of alleviating cachectic muscle wasting. However, as it stands, these aspects of the study remains rather preliminary. This is particularly the case regarding the role of dietary interventions in the model and understanding of the type of metabolic reprogramming in wasting muscles, which lack direct experimental evidence. If the authors were able to further develop this aspects of the study with robust experimental work, it will make it a very valuable and impactful report.

  5. Version published to 10.1101/2023.06.23.546217v1 on bioRxiv