An intestinal Sir2-HSF1-ATGL1 pathway regulates lipolysis in C. elegans

  1. Milán Somogyvári
  2. Saba Khatatneh
  3. Gábor Hajdú
  4. Bar Sotil
  5. József Murányi
  6. Csaba Sőti

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Abstract

Proteostasis maintenance and lipid metabolism are critical for survival and promote longevity, however, their coordination is largely unclear. Here we show that the heat shock factor HSF-1 and the proteostasis state regulates lipolysis in C. elegans . We find that in response to starvation, the sirtuin 1 ortholog SIR-2.1 activates lipolysis by upregulation of the adipose triglyceride lipase ATGL-1. In feeding worms, intestinal HSF-1 represses ATGL-1 expression and lipolysis via the microRNA system. In starving worms, SIR-2.1 suspends a miR-53- mediated suppression of lipolysis by inhibiting its HSF-1-dependent expression. The apparent antagonism of SIR-2.1 and HSF-1, distinct from their synergism at heat shock promoters suggests a context-specific regulation of HSF-1 by SIR-2.1. We demonstrate that the SIR-2.1 and protein kinase A pathways are both indispensable, and independently converge on ATGL- 1 for lipolysis. HSF-1 activation by proteostasis disturbances inhibits starvation-induced lipid mobilization, whereas its age-related decline limits fat deposition through atgl-1 . Our findings reveal a crosstalk between proteostasis and lipid/energy metabolism, which may modulate stress resilience and aging.

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  1. Review Commons

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    Reply to the reviewers

    Manuscript number: RC-2024-02470

    Corresponding author(s): Milán, Somogyvári; Csaba, Sőti

    1. General Statements

    We thank both Reviewers for their constructive comments, and we hope that our reply clarifies the concerns and the revised manuscript will be recommended for publication in Review Commons affiliated journals.

    2. Description of the planned revisions

    As the first major concern, Reviewer #1 raised the question of resolving the link between SIR-2.1 and HSF-1.

    In order to address this issue, we plan to utilize a two-way approach:

    • We plan to check the acetylation status of C. elegans HSF-1 using Mass Spectrometry.
    • We aim to evaluate changes in its promoter binding by utilizing a form of Chromatin Immuno-Precipitation combined with RT-qPCR.
    • As an alternative approach, we plan to utilize the cbp-1GoF mutant strain MH2430, that was described to acetylate HSF-1 and therefore change its transactivational function (Barrett & Westerheide, 2022). We'd like to see whether this can achieve a phenocopy of the sir-2.1(null) genetic background - on the level of the lipolysis phenotype and HSF-1-acetylation. These experiments are to be performed in both wildtype and sir-2.1-silenced animals under fed and starved conditions.

    The second issue raised by Reviewer #1 was to confirm that the miR-53 activity on atgl-1 3' UTR is crucial for the described phenotype.

    Our planned solution for this issue is to create novel C. elegans strains that expresses green fluorescent protein under the regulation of the atgl-1-promoter and with either the wildtype 3'UTR of atgl-1 attached or a mutated one, to which mir-53-binding is not possible. Through fluorescence microscopy experiments involving fed and starved animals, we hope to be able to sufficiently assess the necessity of mir-53 activity to changes in atgl-1 expression and function.* *

    The following minor concern of Reviewer #1 regarding the Results section are addressed here:

    (b) Supporting our ORO results with using the transgene idrIs1[dhs-3p::dhs-3::GFP] to label lipid droplets.

    After acquiring the LIU1 strain harboring the aforementioned transgene, we plan to validate some of the results gained from ORO experiments.

    Reviewer #2 brought into our attention several concerns that need to be addressed:

    1- Firstly, it was mentioned that the overabundance of histograms and the lack of indications on the representative images makes it difficult for the reader to assess the information on the panels.

    We plan to include more images of stained worms while also indicating the changes that the histograms are meant to show. We hope these efforts will make our results more convincing.

    2- Reviewer #2 mentioned that some of the results shown in our manuscript was already published in Zaarur N et al, 2019.

    We thank the Reviewer for bringing this issue to our attention. We'd like to however point out that the mentioned paper by Zaarur et al. is indeed referenced in two places in the Introduction of our manuscript: first, when highlighting that longevity pathways, fat metabolism and lifespan determination are interconnected (line 56), and second, indicating that ATGL-1 mediates longevity in response to dietary restriction and reduced insulin-like signaling (line 65). The regulation of ATGL-1 by starvation and of lipid mobilization by ATGL-1 are not among the novel results of this study. The novelty of our data lies mainly in HSF-1 being involved through specific microRNAs - which to the best of our knowledge has not yet been published. We agree with Reviewer #2 that the visual representation of the data in Zaarur et al. is more pleasing, therefore we plan to incorporate representative images here as well.

    5- Selection of mircoRNA genes

    It was rightly pointed out by Reviewer #2 that mir-53 was reported to be upregulated by HSF-1 upon heat-shock, while there's no mention of it behaving so upon starvation. However, we considered examining it a potentially worthwhile direction given that in C. elegans it is a common observation that various stresses lead to the activation of similar/overlapping stress-response pathways. As seen at Figure 5E, our data supports the idea that mir-53 expression responds to starvation, as its pre-miRNA levels are elevated by starvation in sir-2.1-silenced animals. As for mir-60 and mir-75, we indeed do not have evidence for them being regulated by HSF-1, however their mutants have been associated with reduced body fat content (Brosnan, Palmer & Zuryn, 2021), thus we considered them also to be potential candidates for the role of mediator in the observed lipolysis phenomenon. We aim to make our reasons for choosing these specific miRs clearer in the manuscript.

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

    In accordance with the minor concerns of Reviewer #1, the following changes have already been made to the manuscript:

    In the Abstract, two sentences were changed: (a) "In starving worms, a SIR-2.1-dependent suppression of specific HSF-1 transcriptional activity leads to the inhibition of lipolysis through mitigating miR-53-expression" & (b) "potential crosstalk" was introduced to the last sentence in order to better reflect the nature of our results.

    In Introduction, the start of the first sentence was changed to "Lipids are a diverse group of cellular constituents".

    In Results, the suggested changes in the C. elegans nomenclature were performed (a), where we substituted "sir-2.1 knockout" with "sir-2.1(null)" and "hsf-1 knockout" with "hsf-1(null)".

    The following concerns of Reviewer #2 are addressed in the text of the transferred manuscript:

    4- Stage of synchronized populations in the starvation protocol.

    We thank the Reviewer for highlighting this issue. The life-stage of the animals should indeed be specified at this particular method. It may have been omitted due to RNAi treatments being described just above, where it is mentioned that L4 animals are washed onto RNAi plates where they spend 2 days. Any starvation protocol starts only after these RNAi treatments, since almost all our experiments include some form of RNAi. Therefore, in any trials that do not have RNAi in them, we still only applied starvation from 2 days after L4 stage - for comparability's sake. This issue is clarified in the revised manuscript.

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

    The third major concern mentioned by Reviewer #1 is the epistatic relationships between our novel regulatory pathway and the KIN-1/PKA.

    We'd like to thank Reviewer #1 for turning our attention towards the issue. We feel that the phosphorylation of ATGL-1 by KIN-1 - most probably on Serine 303 - was well established in Lee JH et al, 2014, as seen at Figure 5B - which, naturally, must occur downstream from any transcriptional regulation done by the SIR-2.1-HSF-1-MIR-53 axis. Nevertheless, it would be interesting to see if a non-phosphorylatable ATGL-1 will not support lipolysis upon starvation even if its mRNA expression is activated - which is something that was not tested for in Lee JH et al, 2014. However, since this was not the main subject of our manuscript - more of an addition to ATGL-1-regulation - while our work focused on the regulatory axis going through HSF-1, we do not consider it crucial to perform further experiments aimed at the KIN-1/PKA-mediated regulation of ATGL-1.

    The following minor concerns of Reviewer #1 regarding the Results section are addressed here:

    (c) Fatty acid profiling in the different experimental conditions.

    Even though we feel the potential significance of such data, it does not fit under the scope of our current study. We feel this to be better fitting for a later, follow-up research project.

    (d) Concern about double RNAi treatment in Figure 2D.

    In such particular experiments, where the nematode strain is already a mutant one (where RNAi can only affect intestinal cells), it would require time-consuming crossings with the sir-2.1 and hsf-1 mutant animals. For this reason, we opted to use double RNAi, since according to literature - as well as to our previous experience - double RNAi can be a reliable method to silence the expression of two genes simultaneously. Since the effectivity of RNAi can be influenced by dosage, we compensated for this by mixing the single RNAi treatments with Empty Vector containing bacteria. The results themselves show the silencing-treatments to be effective - to a similar extent as the single RNAi treatments seen in Figure 2A.

    (e) Improving statistical power.

    We agree with Reviewer #1 that in some cases the addition of biological replicates may have the potential to strengthen our conclusions. We argue however, that even though in each case we applied statistical post-hoc tests in order to avoid a type I error, going above the customary 3 biological replicates at each and every experiment would increase the probability of such an error occurring.

    The following concerns of Reviewer #2 are addressed here:

    3- Reviewer #2 inquired about RNAi efficiency and the usage of knock-out mutants.

    Here, we'd like to highlight that throughout the manuscript we utilized sir-2.1(null) mutants in Fig. 1A-B, Fig. 2A-B, Fig. 3D & Fig. S3A; while using hsf-1(null) mutants in Fig. 2A, Fig. 3B & Fig. 8D-F - among other mutants and transgenics. The list of strains can be found in Supplementary Table 1. Regarding the hsf-1(RNAi), it is a strain used for silencing hsf-1-expression reliably in the past by the lab of origin at ELTE University (Barna et al. 2012 BMC Developmental Biology). The silencing of hsf-1 does not lead to any noticeable phenotypes, but it does fully eliminate hsp-70-induction by heat-shock or starvation as shown on Fig. 5A-B.

    *6- *KIN-1 & KIN-2's role and place in lipolysis regulation

    As Reviewer #2 pointed out, both a mutation in kin-1 and kin-2 seemingly lead to the inhibition of lipolysis. However, since kin-2 codes for the regulatory subunit of KIN-1/PKA, a Loss of Function mutation in it leads to a constitutively active PKA - which in turn is expected (among other outcomes) to continuously phosphorylate and thus stabilize ATGL-1. In accordance with this, loss of KIN-1 resulted in an inability to utilize lipid-reserves - therefore the ORO staining levels of these mutants remained similar to wildtype and fed state even upon starvation - while loss of KIN-2 lead to a significantly decreased basal lipid staining, that could not be further decreased by starvation. We argue that the lack of any effect of sir-2.1(RNAi) (or hsf-1(RNAi)) on these phenotypes, while atgl-1(RNAi) was able to revert the kin-2(null)-related basal lipid loss, strongly supports the epistatic relationships proposed.

  2. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    Review

    An intestinal Sir2-HSF1-ATGL1 pathway regulates lipolysis in C. elegans

    In the manuscript by Somogyvári et al., the authors focus on the differences between the fed and the fasted state using C. elegans. In particular the authors find that in the fasted state, the C. elegans SIRT1 ortholog, SIR-2.1, activates lipolysis by upregulation of ATGL-1. Further studies show that in fed worms regulation occurs in the intestine by HSF-1, ATGL-1, and the microRNA system. In contrast, in fasted worms, SIR-2.1 functions with the miR-53 microRNA to affect lipolysis and hsf-1. Further experiments attempt to implicate protein kinase A and proteostasis. Ultimately, the authors attempt to invoke a model for stress resilience and aging. Overall, the data as presented is not very convincing. All of the data is presented as histograms which show only mild effects. Images that are shown are not convincing. Some of the data has been previously published (mentioned below). Therefore, the manuscript needs extensive revisions prior to resubmission and should address the comments below.

    1. why is most of the data presented as a histogram?

    Why are there not representative images that help readers examine the results? /

    For example figure 1A does not really show anything but could guide the reader. The worm images throughout the manuscript do not give any indication of what the authors want the data to show the reader.

    1. some of the data has already been published.

    Mol Metab. 2019 Sep:27:75-82. doi: 10.1016/j.molmet.2019.07.001. Epub 2019 Jul 5. Nava Zaarur et al. fig 1

    ' ATGL-1 is up-regulated by fasting of C. elegans. (A) Wild type (N2) and atgl-1::gfp worms w

    control (Fed) and Fasted groups and stained with Oil Red O.
    (B) Triglyceride content was measured in Fed and Fasted groups of N2 and atgl-1::gfp worms. (C) RNA was extracted from Fed and Fasted groups, and atgl-1 mRNA levels were measured by qRT-PCR; actin-1/3 was used for normalization. (D) Fed and Fasted L4 stage atgl-1::gfp worms were visualized by fluorescence microscopy (200X, equal exposure times). Bar e 50 mM. (E) Quantification of the results shown in panel D by ImageJ (10 randomly selected worms per group). '

    this is not referenced or discussed and more convincing than simply a histogram

      • why is there no analysis with mutants and simply Rnai? for example why is sir2 mutant not used.?
      • does the rnai show any phenotypes? ex hsf-1 rnai = hsf mutant?
      • Do you know the knockdown efficiency for the rnai clones?
    2. Starvation protocol

    560 Synchronized populations were washed 3 times with M9 buffer and placed either on 561 plates containing bacterial food source, or empty plates for 18 hours.

    - what stage were the Synchronized populations?
      1. Brunquell, J., Snyder, A., Cheng, F. & Westerheide, S. D. HSF-1 is a regulator of 699 miRNA expression in Caenorhabditis elegans. PLoS One 12, 1-24 (2017) This is the reference used to define the connection to micrornas. However, this manuscript describes miRNAs induced by heat shock. How does heat shock connect to starvation? The fed or the fasted state? Overall, the rationale for the specific microRNAs shown in the manuscript example mir-53 is unclear.

    2. Figure 6. The protein kinase A KIN-1 affects lipolysis and ATGL-1 function 330 downstream from SIR-2.1 and HSF-1.

      • there is no difference between kin-1 knockout and Kin-2 knock out-so how does one say that it is only Kin-1?
      • where are the differences between fig 6b and 6c?
      • 'The complete 306 inhibition of lipolysis in the absence of sir-2.1 or kin-1 suggests that Sir2 and PKA pathways 307 are equally indispensable and cooperate in lipolysis regulation in the wildtype'
      • does data really show this? ?- not much difference between kin-1 and kin-2- can you really separate the requirements?

    Significance

    Overall, the data as presented is not very convincing. All of the data is presented as histograms which show only mild effects. Images that are shown are not convincing. Some of the data has been previously published (mentioned below). Therefore, the manuscript needs extensive revisions prior to resubmission and should address the comments below.

  3. Review Commons

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

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    Referee #1

    Evidence, reproducibility and clarity

    Summary.

    This study elucidates the contribution of sirtuin 1 ortholog SIR-2.1 in lipid mobilization upon starvation in the nematode Caenorhabditis elegans. The authors claim that HSF-1 controls the expression of adipose triglyceride lipase ATGL-1 in C. elegans gut. Furthermore, they show that SIR-2.1 modulates ATGL-1 activity by regulating the expression of microRNA miR-53 in an HSF-1-dependent manner. The manuscript also describes the interplay between SIR-2.1/HSF-1 and protein kinase A (KIN-1/PKA) in modulating ATGL-1 activity and proteostasis. Finally, the authors claim that lipid mobilization correlates with HSF-1-dependent proteostasis according to the feeding state of the organism.

    Major concerns.

    This manuscript consists of at least three parts that are relatively connected:

    • The impact of SIR-2.1 deficiency on ATGL-1 expression. Here, the main novelty is that HSF-1-dependent regulation of miR-53 defines atgl-1 expression during starvation.
    • The contribution of KIN-1/PKA in lipid mobilization and ATGL-1 activity downstream SIR-2.1 and HSF-1. This link was partially described in a few previous studies (e.g., Lee JH et al, 2014).
    • The contribution of HSF-1 in intestinal proteotoxic stress and fat metabolism. The role of HSF-1 in proteostasis has been well documented, whereas its participation to lipid metabolism has been described in a few studies (e.g., Oleson BJ, 2024).

    The authors provide novel findings that give a better picture of the signaling cascade regulating these biological processes.

    1. However, this study does not conclusively resolve the link between SIR-2.1 and HSF-1. Does Sirtuin 1 influence HSF-1 through histone deacetylation and, therefore, HSF-1 deposition to target genes? Or does Sirtuin regulate HSF-1 acetylation state and therefore its activity? The authors attempted to address these questions with some experiments (Figure 5), however the data are indirect evidence.
    2. Furthermore, microRNAs have multiple targets with various biological functions. Although the authors provide first line of evidence demonstrating the impact of miR-53 on starvation-induced lipolysis, it may be important to confirm that the miR-53 activity on atgl-1 3' UTR is crucial for the described phenotype. Thus, the authors may consider to generate C. elegans strains carrying an atgl-1 3' UTR that is not recognized by the endogenous miR-53 (OPTIONAL).
    3. The role of KIN-1/PKA and ATGL-1 was previously reported (Lee JH et al, 2014) as mentioned in the manuscript. In the submitted manuscript, the authors tried to link KIN-1/PKA, ATGL-1, SIR-2.1 and HSF-1. The authors suggest that "KIN-1 acts downstream from the SIR-2.1 pathway" and "KIN-1 acts downstream of ATGL-1 post-transcriptional regulation". Most of the authors' conclusions are based on RNAi experiments. Could the authors support their claims by providing evidence that the downstream substrates are differentially posttranslationally modified according to the experimental conditions (starvation vs feeding)? Apart from RNAi methods, could the authors support their claims by using non-phosphorylatable ATGL-1 mutants?

    Minor concerns.

    Abstract.

    (a) "SIR-2.1 suspends a miR-53-mediated suppression...". Please adjust the text to make it more understandable.

    (b) "Our findings reveal a crosstalk between proteostasis and lipid/energy metabolism, which may modulate stress resilience and aging.". Which evidence do the authors have that this newly identified crosstalk influences aging? Figure 8 is not sufficient to make such a strong claim.

    Introduction.

    I would encourage the authors to re-word some of their sentences. For example, "lipids are diverse constitutes" sounds strange.

    Results.

    (a) Please, keep in mind the internationally accepted C. elegans nomenclature. For example, substitute "sir-2.1 knockout" with "sir-2.1(null)".

    (b) The authors used Oil Red O (ORO staining) to assess lipid content in nematodes. However, the method has a few limitations and the accurate assessments of fat stores may be variable across experiments. One option is that the authors corroborate their findings with another approach. For example, they may consider to use the transgene idrIs1[dhs-3p::dhs-3::GFP] to label lipid droplets in intestinal cells.

    (c) The authors assessed free-fatty acid content in fed and starved animals. It may be informative to report the individual fatty acid molecules that are mobilized in the different experimental conditions.

    (d) It is always difficult to obtain reproducible results by using two RNAi clones (Figure 2D). The authors should corroborate their results with sir-2.1 and hsf-1 mutant worms.

    (e) For some of the experiments, the statistics may be improved. Since some panels show tendency towards statistical significance (e.g., 8F), it may be important that the authors strengthen their analyses with additional biological replicates. This would help to consolidate their findings and conclusions.

    Significance

    This study reports how Sirtuin 1 can modulate ATGL-1 expression by regulating a microRNA (miR-53). It remains unclear if it is through a direct interaction or via epigenetic remodelling of histone acetylation of target genes. By building up on previous studies, the authors provide additional molecular players that take part in lipid mobilisation during starvation.. The audience can be defined as "specialised" and "basic research".

    My fields of expertise are: metabolism, aging and epigenetics. I work with mice and C. elegans.

  4. Version published to 10.1101/2024.04.11.588856 on bioRxiv