Cellular metabolic reprogramming controls sugar appetite in Drosophila

Abstract

Cellular metabolic reprogramming is an important mechanism by which cells rewire their metabolism to promote proliferation and cell growth. This process has been mostly studied in the context of tumorigenesis, but less is known about its relevance for nonpathological processes and how it affects whole-animal physiology. Here, we show that metabolic reprogramming in Drosophila female germline cells affects nutrient preferences of animals. Egg production depends on the upregulation of the activity of the pentose phosphate pathway in the germline, which also specifically increases the animal’s appetite for sugar, the key nutrient fuelling this metabolic pathway. We provide functional evidence that the germline alters sugar appetite by regulating the expression of the fat-body-secreted satiety factor Fit. Our findings demonstrate that the cellular metabolic program of a small set of cells is able to increase the animal’s preference for specific nutrients through inter-organ communication to promote specific metabolic and cellular outcomes.

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Fig. 1: The germline undergoes a reprogramming of its carbohydrate metabolism, which is required for egg production.
Fig. 2: Dietary supply of sugars is required for egg production.
Fig. 3: Carbohydrate metabolism in a subset of germline cells modulates sucrose appetite.
Fig. 4: Germline-ablated females do not have increased available carbohydrates and are in a hyper-starved state.
Fig. 5: The activity of the PPP in the germline is required for egg production.
Fig. 6: PPP activity in the germline modulates sucrose appetite.
Fig. 7: The germline modulates sugar appetite by regulating the expression of the fat-body-secreted satiety peptide Fit.

Data availability

The authors declare that the main data supporting the findings of this study are available within the article and its Supplementary Information files. The detailed genotypes of the animals used in this study are included in Supplementary Tables 1 and 2. Sequences of all oligonucleotides used in this study are included in Supplementary Table 4. The exonic DNA sequences of Hex-A and Pgd were retrieved from Ensembl genome browser 93 (https://www.ensembl.org/index.html) for synthesizing the probes for in situ hybridization. Source imaging data were deposited in figshare (https://doi.org/10.6084/m9.figshare.12600185.v2). Source data are provided with this paper.

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Acknowledgements

We thank B. Dickson (Howard Hughes Medical Institute, Janelia Research Campus, USA), R. Neumüller (Institute of Molecular Biotechnology, Austria), D. McKearin (Howard Hughes Medical Institute, University of Minnesota, USA), M. Buszczak (University of Texas Southwestern Medical Center, USA), Y. Li (Institute of Biophysics, Chinese Academy of Sciences, China), R. Martinho (Instituto de Biomedicina, Universidade de Aveiro, Portugal) and P. Prudencio (Instituto de Medicina Molecular, Portugal) for providing fly stocks and reagents. Lines obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537) were used in this study. We thank R. Martinho and P. Prudencio for help with experimental protocol optimization. We thank R. Martinho, C. Pereira, A. Gontijo, S. Walker, G. Ezra, D. Goldschmidt, P. Francisco, D. Münch and all members of the Behavior and Metabolism Laboratory for helpful discussions and comments on the manuscript and G. Costa for illustrations. We thank M. Anjos and N. Archer for technical assistance. This project was supported by Bial grant 279/16 and Portuguese Foundation for Science and Technology (FCT) postdoctoral fellowship SFRH/BPD/79325/2011 to Z.C.-S. Work by Z.C.-S. was also financed by national funds through the FCT in the framework of the financing of the Norma Transitória DL 57/2016. Work by R.C.-F. was financed by the FCT doctoral fellowship SFRH/BD/143862/2019. Work by I.T. was financed by Marie Skłodowska-Curie Actions postdoctoral fellowship MSCA-IF-EF-ST 867459. Research at the Centre for the Unknown is supported by the Champalimaud Foundation and the research infrastructure Congento, co-financed by Lisboa Regional Operational Programme (Lisboa2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) and Fundação para a Ciência e Tecnologia (Portugal) under the project LISBOA-01-0145-FEDER-022170.

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Contributions

C.R. and Z.C.-S. conceived and designed the experiments. Z.C.-S., R.C.-F., A.P.E. and C.B. performed the experiments. C.R., Z.C.-S., R.C.-F., A.P.E., I.T. and C.B. analysed the data. C.R. and Z.C.-S. wrote the paper. C.R. administered the project. C.R. acquired funding. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Zita Carvalho-Santos or Carlos Ribeiro.

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The authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Related to Figs. 1, 3, 5, and 6. Expression of shRNAs for genes encoding carbohydrate metabolism enzymes in the female germline leads to efficient mRNA knockdown.

Normalized Hex-A (a), Pgi (b), Pfk (c), PyK (d), and Rpi (e) mRNA levels in females fed on holidic medium lacking sucrose. The MTD-GAL4 was used to drive shRNAs in the germline and matching GFP knockdown lines were used as controls (light orange, controls; dark orange, females with PPP knocked down germlines). Grey circles represent independent measurements (n), and the line represents the mean and the error bars the s.e.m. Black filled circles represent the presence and open black circles the absence of a given dietary nutrient or transgene. Full genotypes can be found in Supplementary Tables 1 and 2. Statistical significance was tested using a One-sided unpaired t-test. Source data

Extended Data Fig. 2 Related to Fig. 3. Germline-ablated virgin and mated females show a specific decrease in sugar appetite.

a, Virgin females were assayed for changes in nutrient choice using the flyPAD. Bars represent the difference in sucrose feeding of flies (light orange, controls; dark orange, germline-ablated) fed on holidic medium lacking sucrose (carb deprived) vs full holidic medium (full). Columns represent the mean and the error bars show 95% confidence interval. Black filled circles represent the presence and open black circles the absence of a transgene. The method used to calculate the plotted values is described in the Methods section. b, Sugar preference was assayed using the red and blue food choice assay of flies (light colours, controls; dark colours, germline-ablated) kept on full holidic medium (green) or medium lacking sucrose (orange). Colored circles in the plot represent sugar preference in single assays, and the line represents the median and error bars the interquartile range. The number of independent biological replicates is represented as n. a,b, Full genotypes can be found in Supplementary Tables 1 and 2. Statistical significance was tested using the Wilcoxon rank-sum test (a) or the Kruskal-Wallis test followed by Dunn’s multiple comparison test (b). Source data

Extended Data Fig. 3 Related to Fig. 4. Germline-ablated females do not have increased levels of trehalose or fructose.

Trehalose (a) or fructose (b) measurements from the heads of females (light colours, controls; dark colours, germline-ablated) reared on full holidic medium (green) or holidic medium lacking sucrose (orange) normalized to protein concentrations. Grey circles represent independent measurements (n), and the line represents the mean and the error bars the s.e.m. Black filled circles represent the presence and open black circles the absence of a given dietary nutrient or transgene. Full genotypes can be found in Supplementary Tables 1 and 2. Statistical significance was tested using an ordinary one-way ANOVA followed by Sidak’s multiple comparisons test. Source data

Extended Data Fig. 4 Related to Fig. 5. The activity of the pentose phosphate pathway in the germline is required for oogenesis.

Ovariole morphology revealed by immunostaining of ovaries from females in which Zw (a) or Pgd (b) was knocked down in the germline. The dashed line in the germaria (G) delimits the region where St1 egg chambers would be formed. 1B1: spectrosomes and fusomes, Phalloidin: actin, DAPI: DNA. Scale bars, 25 μm (Germarium) and 50 μm (Ovariole). Full genotypes can be found in Supplementary Tables 1 and 2. Data shown are representative of 2 independent experiments of at least 6 ovaries/condition.

Extended Data Fig. 5 Related to Fig. 6. Knockdown of Hex-A, Pgd, or Rpi in ovaries using a mid-oogenesis driver leads to changes in sugar appetite.

a-c, Females in which the germline was knockdown of PPP enzymes (dark orange) using a mid-oogenesis driver (matα4-GAL4) were assayed for an effect in nutrient choice using the flyPAD. Bars represent the difference in sucrose feeding of flies maintained on holidic medium lacking sucrose (carb deprived) vs full holidic medium (full). Genotype matched GFP knockdown lines were used as negative controls (light orange). Columns represent the mean and the error bars show 95% confidence interval. Black filled circles represent the presence and open black circles the absence of a given transgene. The raw data used to derive these plots as well as the number of individuals tested per condition is indicated in Supplementary Fig. 2. The method used to calculate the plotted values is described in the Methods section. Statistical significance was tested using the Wilcoxon rank-sum test. d, Ovariole morphology revealed by immunostaining of ovaries from females in which Pgd was knocked down in the germline using matα4-GAL4 and the corresponding negative control. The region where the germarium is localized within the ovariole is marked with a G. Phalloidin: actin, DAPI: DNA. Scale bars, 25 μm (Germarium) and 50 μm (Ovariole). Data shown are representative of 1 experiment of at least 6 ovaries/condition. a-d, Full genotypes can be found in Supplementary Tables 1 and 2. Source data

Extended Data Fig. 6 Related to Fig. 6. PPP activity in the germline is required and sufficient for controlling sugar appetite.

a, Schematic depicting the model of how the knockdown of different PPP enzymes may lead to opposite metabolic and behavioral outcomes. Enzymes or metabolites depicted in red are knocked down or decreased while those represented in green accumulate. b, Circles represent the nº of eggs laid/female in single assays (n) and the line represents the mean. The MTD-GAL4 was used to drive shRNAs in the germline and GFP knockdown used as a negative control (light green). Black filled circles represent the presence and open black circles the absence of a transgene. Statistical significance was tested using a One-sided unpaired t-test. Ovariole morphology revealed by immunostaining of ovaries from females in which Rpi was knocked down in the germline (c) or were mutants for Pgd and Zw (d). The region where the germarium is localized within the ovariole is marked with a G. 1B1: spectrosomes and fusomes, Phalloidin: actin, DAPI: DNA. Scale bars, 25 μm (Germarium) and 50 μm (Ovariole). Full genotypes can be found in Supplementary Tables 1 and 2. Data shown are representative of 2 experiments of at least 6 ovaries/condition. Source data

Extended Data Fig. 7 Females with severely affected germline induced by AA deprivation still strongly increase sucrose appetite upon sugar deprivation.

a, Wild type females were assayed for an effect in nutrient choice using the flyPAD after being fed on a complete holidic medium, a medium lacking sucrose or a medium lacking both AAs and sucrose. Bars represent the difference in sucrose feeding of flies maintained on holidic medium lacking sucrose or sucrose and AAs (deprived) vs full holidic medium (full) (orange vs grey). Columns represent the mean and the error bars show 95% confidence interval. Raw data used to generate these plots and the number of individuals tested/condition is indicated in (b). The method used to calculate the plotted values is described in the Methods section. The raw data in (b) is represented by boxes showing the median with upper/lower quartiles. The number of independent biological replicates is represented as n. a, b, Black filled circles represent the presence and open black circles the absence of a particular nutrient (green, full HM, orange, HM without sucrose; grey, HM without both AAs and sucrose). Full genotypes can be found in Supplementary Tables 1 and 2. Statistical significance was tested using the Wilcoxon rank-sum test (a) the One-sided Mann-Whitney test (b). Source data

Extended Data Fig. 8 Related to Fig. 7. Carbohydrate metabolism in the germline controls the expression levels of fit.

a, Normalized fit mRNA levels in females fed on holidic medium lacking sucrose. The MTD-GAL4 was used to drive shRNA in the germline. A genotype matched GFP knockdown line was used as a control (light orange). b, Normalized fit mRNA levels in control females fed on a complete holidic medium (green) or a medium lacking sucrose (orange). a-b, Grey circles represent independent measurements (n), and the line represents the mean and the error bars the s.e.m. c, Females (light colours, controls; dark colours, females mutant for fit) were assayed for an effect in nutrient choice using the flyPAD after fed on a complete holidic medium (green), or one lacking sucrose (orange). Boxes represent median with upper/lower quartiles. The number of independent biological replicates is represented as n. a-c, Black filled circles represent the presence and open black circles the absence of a nutrient, transgene, the germline, or the fit gene. Full genotypes can be found in Supplementary Tables 1 and 2. Statistical significance was tested using the One-sided unpaired t-test (a-b) or the Kruskal-Wallis test followed by Dunn’s multiple comparison test (c). Source data

Extended Data Fig. 9 The male germline also regulates carbohydrate appetite but not via the PPP.

Males in which the germline was ablated (nos-GAL4>UAS-bam) (a) or metabolically manipulated (MTD-GAL4>Zw shRNA, MTD-GAL4>Pgd shRNA) (b) were assayed for an effect in nutrient choice using the flyPAD. Bars represent the difference in sucrose feeding of flies (light orange, controls; dark orange, germline-ablated) maintained on holidic medium lacking sucrose (carb deprived) vs full holidic medium (full). Genotype matched GFP knockdown lines were used as negative controls in (b). Columns (light orange, controls; dark orange, males with PPP knocked down germlines) represent the mean and the error bars show 95% confidence interval. Black filled circles represent the presence and open black circles the absence of a transgene. The raw data used to derive these plots as well as the number of individuals tested per condition is indicated in Supplementary Fig. 3. The method used to calculate the plotted values is described in the Methods section. Full genotypes can be found in Supplementary Tables 1 and 2. Statistical significance was tested using the Wilcoxon rank-sum test. Source data

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Carvalho-Santos, Z., Cardoso-Figueiredo, R., Elias, A.P. et al. Cellular metabolic reprogramming controls sugar appetite in Drosophila. Nat Metab 2, 958–973 (2020). https://doi.org/10.1038/s42255-020-0266-x

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