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A developmental checkpoint directs metabolic remodelling as a strategy against starvation in Drosophila

Nature Metabolism volume 2, pages 1096–1112 (2020)Cite this article

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Abstract

Steroid hormones are crucial regulators of life-stage transitions during development in animals. However, the molecular mechanisms by which developmental transition through these stages is coupled with optimal metabolic homeostasis remains poorly understood. Here, we demonstrate through mathematical modelling and experimental validation that ecdysteroid-induced metabolic remodelling from resource consumption to conservation can be a successful life-history strategy to maximize fitness in Drosophila larvae in a fluctuating environment. Specifically, the ecdysteroid-inducible protein ImpL2 protects against hydrolysis of circulating trehalose following pupal commitment in larvae. Stored glycogen and triglycerides in the fat body are also conserved, even under fasting conditions. Moreover, pupal commitment dictates reduced energy expenditure upon starvation to maintain available resources, thus negotiating trade-offs in resource allocation at the physiological and behavioural levels. The optimal stage-specific metabolic shift elucidated by our predictive and empirical approaches reveals that Drosophila has developed a highly controlled system for ensuring robust development that may be conserved among higher-order organisms in response to intrinsic and extrinsic cues.

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Fig. 1: Optimal control theory predicts a life-history strategy in Drosophila.
Fig. 2: Resource storage in larvae contributes to adult fitness over metamorphosis.
Fig. 3: Global changes in metabolism during the last larval instar and metamorphosis.
Fig. 4: Glycogen and stored lipids in the fat body are conserved after pupal commitment.
Fig. 5: Metabolic remodelling in the fat body is directed by ecdysteroid signalling.
Fig. 6: Ecdysteroid-inducible ImpL2 is required for the maintenance of trehalose.
Fig. 7: ImpL2 impacts fitness costs through inhibition of Treh.
Fig. 8: Larvae after CW exhibit reduced metabolic activity following starvation.

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Data availability

The data supporting the findings of this study are available within the article and its Supplementary Information. Sequences of oligonucleotides used in this study are included in Supplementary Table 1. Source data are provided with this paper.

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Acknowledgements

We thank O. Baba (Tokushima University), M.B. O’Conner (University of Minnesota), H. Sasaki (Osaka University), H. Stocker (ETH Zürich), the Bloomington Drosophila Stock Center, the National Institute of Genetics Drosophila Stock Center and the Kyoto Drosophila Stock Center for fly stocks and antibodies; members of fly laboratories in RIKEN BDR for their valuable support and discussion; the genome resource and analysis unit in RIKEN BDR for their assistance with Sanger sequencing; R. Nakagawa for performing protein identification by MS analysis; W. Kimura for providing laboratory equipment; Y. Shimada and R. Niwa for helpful comments and discussions on the manuscript. This work was supported in part by the Japan Society for the Promotion of Science (JSPS) KAKENHI grant nos. 17H03658 and 17K19433 (to T.N.) and by Grant-in-Aid for JSPS Fellows no. 15J04931 (to K.H.).

Author information

Author notes

  1. Ken-ichi Hironaka

    Present address: Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo, Japan

  2. These authors contributed equally: Takayuki Yamada, Ken-ichi Hironaka.

Authors and Affiliations

  1. Laboratory for Growth Control Signaling, RIKEN Center for Biosystems Dynamics Research (BDR), Kobe, Japan

    Takayuki Yamada, Ken-ichi Hironaka, Okiko Habara & Takashi Nishimura

  2. Laboratory for Developmental Morphogeometry, RIKEN BDR, Kobe, Japan

    Yoshihiro Morishita

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Contributions

T.Y., K.H. and T.N. designed the experiments. T.Y., H.O. and T.N. performed the experiments. T.Y., K.H. and T.N. performed the data analysis. K.H. and Y.M. conducted the mathematical modelling. K.H. and T.N. wrote the manuscript. All authors discussed and reviewed the manuscript.

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Correspondence to Takashi Nishimura.

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Extended data

Extended Data Fig. 1 Parameter dependence of the optimal policy for pre-CW and post-CW larvae.

a, Parameter dependency of pre-CW larvae. The x and y axes indicate the time t and stored resources S, respectively. The optimal policy of pre-CW larvae was examined in the subspace of parameter space. Red and blue arrows indicate the optimal policy at the feeding state (x = 1) and fasting state (x = 0), respectively. Whereas the optimal behaviour at the fasting state is always more or less release, that of the feeding state is not always storage. Even in the feeding state, the animal should release the stored resources (i) when the resource requirements for survival are too high (that is, both μmax and \(K_{\hat A}\) are large) or (ii) when the environment is stable enough not to require storage (that is, both PE and τE are large). As a result, the animal would never store resources inside its body. Therefore, the corresponding parameter regions are biologically implausible for many animals that store resources. b, Typical optimal policy of post-CW larvae at the beginning (t = 0). c, Three types of functional forms of terminal reward: linear, step, and ramp. d, Parameter dependency of the optimal policy in post-CW larvae when using the linear function as ψ. The saving behaviour appears immediately before T in any parameter regions when using the same parameter subspaces as in (a). However, the area of saving behavior is dependent on the parameters. In particular, when the life extension by resource consumption is very effective (μmax is large and \(K_{\hat A}\) is small), the saving area becomes narrow. e, Parameter dependency of the optimal policy in post-CW larvae when using the step function as ψ (θ =0.5). The saving behavior no longer occurs at \(\hat S \ge \theta\) (particularly noticeable at the rightmost column, just before Tpost). Such policy is intuitively natural: because the excessively stored resource above the threshold provides no added benefit in later life, the resource should be consumed to increase survival rate. f, Parameter dependency of the optimal policy in post-CW larvae when using the ramp function as ψ (θ= 0.5). The saving behavior occurs immediately before Tpost for all \(\hat S\). Thus, depending on the functional form of terminal reward ψ, the optimal policy can vary. For example, if the focal animal has the maximum storage resources and shows the saving behavior, the terminal reward function is unlikely to be stepwise. In our case, fly larvae were reared for several days in the experimental condition with sufficient nutrition and thus can be assumed to have reached maximum storage. When artificially transferred to a starvation medium, such well-fed larvae showed saving behavior, suggesting that the terminal reward is not considered step-like. Therefore, even if the terminal reward has a threshold θ, the excess resource storage above the threshold \(\left( {\hat S - \theta } \right)\) will bring some fitness advantage to animals (for example, ramp function), such as the increase in the number of eggs and the survival rate in the adult stage.

Extended Data Fig. 2 Parameter dependency of the optimal enzyme activity and metabolic flux.

a,b, Parameter dependency of the optimal enzyme activity and metabolic flux in the pre-CW larvae (a) and post-CW larvae with the linear terminal reward (b). The x and y axes indicate the time t and stored resources S, respectively. Here the optimal policy of storage activity kstorage, release activity krelease, storage flux vstorage, and release flux vrelease were examined. Circles and arrows indicate the magnitude of activities (kstorage or krelease) and fluxes (vstorage or vrelease), respectively. Red and blue colors mean the optimal policy at the feeding state (x = 1) and fasting state (x = 0), respectively. For the post-CW larvae, the policies before the endpoint (t=Tpost) are shown.

Extended Data Fig. 3 Parameter dependency of the optimal enzyme activity and metabolic flux.

a,b, Parameter dependency of the optimal enzyme activity and metabolic flux in the post-CW larvae with the step terminal reward (a) and with the ramp terminal reward (b). The x and y axes indicate the time t and stored resources S, respectively. The same threshold parameters are used in both cases (θ = 0.5). Circles and arrows indicate the magnitude of activities (kstorage or krelease) and fluxes (vstorage or vrelease), respectively. Red and blue colors mean the optimal policy at the feeding state (x = 1) and fasting state (x = 0), respectively. The policies before the endpoint (t=Tpost) are shown.

Extended Data Fig. 4 Global changes in metabolism during the last larval instar and metamorphosis.

a, Sampling scheme for metabolomics analysis. Control (w-) early third-instar larvae grown on normal food (L0) were transferred and cultured on either normal food (fed) or agar food (starved) for the indicated times. ALH, after larval hatching; CW, critical weight; P0, white pupae. Asterisks indicate pupal samples in which larvae were starved after reaching CW. b, Changes in amounts of total protein per animal. Pink and yellow regions indicate prepupal and pupal stages, respectively. c, Principal component (PC) analysis of metabolites in the larval or the pupal period. Ellipses of clusters show the 95% confidence regions for each sample group. d, Hierarchical clustering analysis and corresponding heat maps were constructed based on the top 50 metabolites ranked by one-way ANOVA. The left and right heat maps indicate metabolite data in strains Oregon-R and w-, respectively. Values shown are means±SEM; n = 3 biologically independent samples, except at L16 (n = 4) and at P0 (n = 6).

Extended Data Fig. 5 Metabolomics analysis during the last larval instar and metamorphosis.

a, Heat maps of selected metabolites under normal rearing conditions in strains Oregon-R and w-. Red and blue indicate increased and decreased metabolites relative to median metabolite levels, respectively. The left and right heat maps indicate metabolite data in Oregon-R and w-, respectively. b, Principal component (PC) analysis of metabolites during the last larval instar and pupal periods in wild-type Oregon-R strain and c, control strain w-. Time points in each sample are indicated to the right. Ellipses of clusters show the 95% confidence regions for each sample group. PC1 represents the larval and pupal periods; white pupae (P0) are located at the intermediate. PC2 discriminates between fed and starved larvae; pupae are located at the intermediate in the direction of PC2. PC4 (PC3 for w-) represents a transient response at pupariation.

Extended Data Fig. 6 Metabolic response to starvation.

a, Overview of glucose and storage sugar metabolism. Genes that function in glycogen and trehalose metabolism are shown in red. b, Maintenance of fat body glycogen after starvation at E3rd+24 h was confirmed by immunostaining with anti-glycogen antibodies. c, Glycogen in the fat body, but not in the body wall muscles, continues to increase during the third instar. The tissue distribution of glycogen was analysed by LC-MS/MS after enzymatic digestion. Amounts of protein and glycogen are shown as values per larva or values normalized to the protein levels in each tissue. CNS, central nervous system. d, Starvation during the second instar did not affect the metabolic changes in post-CW larvae. Control early second instar (E2nd) larvae grown on normal diet (ND) were transferred and cultured on agar for 24 h, transferred to ND, and then transferred again on agar for 12 h at the indicated stages (bottom). Fat body glycogen was visualized by PAS staining at the indicated time points (left). Relative amounts of trehalose are shown (right). The x-axis indicates the time at which the animals were switched to starvation. e, Relative intensity of Oil Red O signal in each oenocyte cluster. n = 4 biologically independent samples (c, d). The number of oenocyte clusters from multiple larvae is represented by n (e). Values shown are means±SD (c,d). In the box plots (e), the centerlines show the medians, the box limits indicate the 25th and 75th percentiles, and the whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Two-tailed Student’s t-test (d), one-way ANOVA with Tukey’s post hoc test (e). Scale bars: 100 μm. The experiments were repeated independently at least twice with similar results (b,d).

Source data

Extended Data Fig. 7 Ecdysteroid-directed metabolic reprogramming.

a, Expression of EcR-DN, but not EcR-RNAi, in the fat body (Cg-Gal4) decreased fat body glycogen at the wandering stage as determined by PAS staining. EcR-DN inhibits both the activation and de-repression functions of EcR because it cannot bind ecdysteroids6,9. In contrast, EcR-RNAi suppresses the function of EcR but induces de-repression, which mimics some effects of ecdysteroids. Therefore, a high concentration of ecdysteroids at the wandering stage appears to protect glycogen via de-repression. b, Quantification of PAS signal in the fat body of the indicated genotypes and time points. c, Inhibition of EcR in the fat body slightly delays pupariation by approximately 10 h. However, when these larvae were starved at E3rd+24 h, we observed pupariation within the following two days, indicating that these larvae have reached CW at E3rd+24 h. Expression of mCherry-RNAi and GlyS-RNAi had no noticeable effect under these conditions. d, Log-fold changes in 20-hydroxyecdysone (20E) levels during the third instar. Grey region indicates the wandering stage for pupariation. e, Effects of oral administration of 20E on gene expression of glycogen metabolism genes (functions shown in Extended Data Fig. 6a). f, Expression of EcR-DN and EcR-RNAi in the fat body had no obvious effect on levels of triglycerides. Lipid droplets were visualized by Oil Red O staining. g, Relative amounts of TAG in whole larvae of the indicated genotypes and conditions. h, Oil Red O staining in oenocytes. Expression of EcR-RNAi, but not EcR-DN, in the fat body induced accumulation of neutral lipids in oenocytes at E3rd+24 h under fed conditions. In contrast, EcR-DN, but not EcR-RNAi, induced lipid accumulation at the wandering stage. EcR likely controls lipid metabolism with distinct mechanisms at these two stages. Of note, inhibition of EcR had no effect at E3rd. Relative intensity of Oil Red O signal in each oenocyte cluster is shown on the bottom. The lipid accumulation in oenocytes did not visibly reflect the lipolysis in the fat body, most likely due to the huge amounts of lipid stores24. The number of fat body clusters (b), oenocyte clusters (h), or biologically independent samples (g) is represented by n and the lines represent the mean (b,h). n = 4 (d), n = 3 (e) biologically independent samples. Values shown are means±SEM (d), means±SD (e,g). Two-tailed Student’s t-test (e,g), two-tailed Kruskal-Wallis test followed by Dunn’s post hoc test (b,h). Scale bars: 100 μm (a), 50 μm (f,h). The experiments were repeated independently at least twice with similar results (f).

Source data

Extended Data Fig. 8 Mechanism of trehalose maintenance in post-CW starved larvae.

a, Temporal knockdown of Tps1 reduced the level of trehalose upon starvation in post-CW larvae. Thus, trehalose synthesis in the fat body plays a role in maintaining trehalose levels in post-CW starved larvae. E3rd or M3rd grown on normal diet at 18°C (0 h) was transferred to a vial containing agar-only diet for 12 h at 30°C (12 h starve), which allows knockdown of Tps1 in the fat body. b, Changes in mRNA expression levels of the trehalose transporters, Tret1–1 and Tret1–2, during the third instar. Tret1–1 is predominantly expressed in the fat body, while Tret1–2 is ubiquitously expressed in various tissues21. c, Full-scan image shown in Fig. 6b. Asterisks and double asterisks indicate non-specific bands and degradation products of cTreh, respectively. d, Tissue distribution of cTreh and sTreh as determined by western blot analysis. e, Protein amounts of samples shown in Fig. 6c were evaluated by SDS-PAGE, followed by CBB staining. f, The interaction of Treh with ImpL2 was confirmed by GST pull-down assay. In addition to ImpL2, we identified Ance-4 and a mixture of larval serum proteins, Lsp2/1α/1β/1γ, by affinity column purification shown in Fig. 6e. Ance-4 is a family gene product of angiotensin-converting enzyme. Treh interacted with Ance-4, while specific interaction was not detected with Lsp2/1α/1β/1γ under these conditions. g, Feeding 20E at E3rd promoted the expression of Lsp2, Lsp1α, and Lsp1γ, while Ance-4 and Lsp1β expression were down-regulated. h, The expression of Ance-4 did not increase during the third instar. According to the results shown in f–h, we focused on ImpL2 for further analysis. i, Purified proteins used in Fig. 6i were evaluated by SDS-PAGE, followed by CBB staining. j, Loss or increase of ImpL2 did not affect the protein amount of sTreh in hemolymph at the indicated stages. k, ImpL2 and SDR mutants increased body growth to the same extent at approximately 20%. Pupal volume with a mixture of male and female are shown. l, Loss of ImpL2 and SDR increased IIS to the same extent at E3rd, as revealed by the down-regulation of Foxo-target genes, InR and 4E-BP (Thor; FlyBase). Values shown are means±SEM (b,h), means±SD (a,g,l). In the box plots (j,k), the centerlines show the medians, the box limits indicate the 25th and 75th percentiles, and the whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. n = 7 (a), n = 3 (g,h), n = 4 (j), n = 6 (l) biologically independent samples. n = 7 biologically independent samples (b), except at 12 h (n = 4). The number of animals is represented by n (k). One-way ANOVA with Tukey’s post hoc test (a,k,l), two-tailed Student’s t-test (g,j). The experiments were repeated independently at least twice with similar results (c–f).

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Extended Data Fig. 9 Validation of larval feeding assay.

a, The amount of ingested normal diet increased in a time- and body size-dependent manner. Feeding assays with ND containing blue dye was performed for the indicated period using larvae at 6 h after starvation. In the violin plots, the white circles show the medians, the box limits indicate the 25th and 75th percentiles, the whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, and the polygons represent density estimates of data and extend to extreme values. The number of biologically independent samples is represented by n.

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Supplementary information

Reporting Summary

Supplementary Tables

Supplementary Table 1

42255_2020_293_MOESM3_ESM.xlsx

Supplementary Dataset 1. Metabolomics profiles of wild-type (Oregon-R) and control (w-) strains. a,b, Metabolomics profiles of wild-type Oregon-R (a) and control w- (b) strains. Values shown are the peak areas, which were normalized to total protein levels after subtraction of blank sample values. Mean and s.d. values are shown. c, Numbers of biological replicates.

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Supplementary Dataset 2 Loading scores of selected metabolites. a, Loading scores for the top 25 metabolites that contributed maximally to PC1/PC2 in Fig. 3c and to PC1/PC2/PC3 in Extended Data Fig. 4c. b, Loading scores for the top 25 metabolites that contributed maximally to PC1/PC2/PC4 in Extended Data Fig. 5b and to PC1/PC2/PC3 in Extended Data Fig. 5c. The most positive and negative loadings are listed separately. Common metabolites between the two strains in each category are marked in red.

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Unprocessed western blots

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Yamada, T., Hironaka, Ki., Habara, O. et al. A developmental checkpoint directs metabolic remodelling as a strategy against starvation in Drosophila. Nat Metab 2, 1096–1112 (2020). https://doi.org/10.1038/s42255-020-00293-4

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