The transcription factor dFOXO controls the expression of insulin pathway genes and lipids content under heat stress in Drosophila melanogaster

The insulin/insulin-like growth factor signaling (IIS) pathway is one of the key elements in an organism’s response to unfavourable conditions. The deep homology of this pathway and its evolutionary conservative role in controlling the carbohydrate and lipid metabolism make it possible to use Drosophila melanogaster for studying its functioning. To identify the properties of interaction of two key IIS pathway components under heat stress in D. melanogaster (the forkhead box O transcription factor (dFOXO) and insulin-like peptide 6 (DILP6), which intermediates the dFOXO signal sent from the fat body to the insulin-producing cells of the brain where DILPs1–5 are synthesized), we analysed the expression of the genes dilp6, dfoxo and insulin-like receptor gene (dInR) in females of strains carrying the hypomorphic mutation dilp6⁴¹ and hypofunctional mutation foxo^BG01018. We found that neither mutation influenced dfoxo expression and its uprise under short-term heat stress, but both of them disrupted the stress response of the dilp6 and dInR genes. To reveal the role of identified disruptions in metabolism control and feeding behaviour, we analysed the effect of the dilp6⁴¹ and foxo^BG01018 mutations on total lipids content and capillary feeding intensity in imago under normal conditions and under short-term heat stress. Both mutations caused an increase in these parameters under normal conditions and prevented decrease in total lipids content following heat stress observed in the control strain. In mutants, feeding intensity was increased under normal conditions; and decreased following short-term heat stress in all studied strains for the first 24 h of observation, and in dilp6⁴¹ strain, for 48 h. Thus, we may conclude that dFOXO takes part in regulating the IIS pathway response to heat stress as well as the changes in lipids content caused by heat stress, and this regulation is mediated by DILP6. At the same time, the feeding behaviour of imago might be controlled by dFOXO and DILP6 under normal conditions, but not under heat stress.

1. Álvarez-Rendón J.P., Salceda R., Riesgo-Escovar J.R. Drosophila melanogaster as a model for diabetes type 2 progression. Biomed. Res. Int. 2018;2018:1417528. DOI 10.1155/2018/1417528.

2. Andreenkova O.V., Eremina M.A., Gruntenko N.E., Rauschenbach I.Y. Effect of heat stress on expression of DILP2 and DILP3 insulin-like peptide genes in Drosophila melanogaster adults. Russ. J. Genet. 2018;54(3):363-365. DOI 10.1134/S102279541803002X.

3. Andreenkova O.V., Rauschenbach I.Y., Gruntenko N.E. Hypomorphic mutation of the dilp6 gene increases DILP3 expression in insulinproducing cells of Drosophila melanogaster. Russ. J. Genet. 2017; 53(10):1159-1161. DOI 10.1134/S1022795417080026.

4. Arrese E.L., Soulages J.L. Insect fat body: energy, metabolism, and regulation. Annu. Rev. Entomol. 2010;55:207-225. DOI 10.1146/annurev-ento-112408-085356.

5. Bai H., Kang P., Tatar M. Drosophila insulin-like peptide-6 (dilp6 ) expression from fat body extends lifespan and represses secretion of Drosophila insulin-like peptide-2 from the brain. Aging Cell. 2012;11(6):978-985. DOI 10.1111/acel.12000.

6. Dionne M.S., Pham L.N., Shirasu-Hiza M., Schneider D.S. Akt and foxo dysregulation contribute to infection-induced wasting in Drosophila. Curr. Biol. 2006;16(20):1977-1985. DOI 10.1016/j.cub.2006.08.052.

7. Eremina M.A., Gruntenko N.E. Adaptation of the sulfophosphovanillin method of analysis of total lipids for various biological objects as exemplified by Drosophila melanogaster. Vavilovskii Zhurnal Genetiki i Selektsii = Vavilov Journal of Genetics and Breeding. 2020; 24(4):441-445. DOI 10.18699/VJ20.636. (in Russian)

8. Eremina M.A., Karpova E.K., Rauschenbach I.Yu., Pirozhkova D.S., Andreenkova O.V., Gruntenko N.E. Mutations in the insulin signaling pathway genes affect carbohydrate level under heat stress in Drosophila melanogaster females. Russ. J. Genet. 2019; 55(4):519-521. DOI 10.1134/S1022795419030050.

9. Even N., Devaud J.M., Barron A.B. General stress responses in the honey bee insects. Insects. 2012;3(4):1271-1298. DOI 10.3390/insects3041271.

10. Garbuz D.G., Evgen’ev M.B. The evolution of heat shock genes and expression patterns of heat shock proteins in the species from temperature contrasting habitats. Russ. J. Genet. 2017;53(1):21-38. DOI 10.1134/S1022795417010069.

11. Gontijo A.M., Garelli A. The biology and evolution of the Dilp8-Lgr3 pathway: a relaxin-like pathway coupling tissue growth and developmental timing control. Mech. Dev. 2018;154:44-50. DOI 10.1016/j.mod.2018.04.005.

12. Gruntenko N.E. Stress and Reproduction of Insects: Hormonal Control. Novosibirsk; Moscow: KMK Publ., 2008. (in Russian)

13. Gruntenko N.E., Adonyeva N.V., Burdina E.V., Karpova E.K., Andreenkova O.V., Gladkikh D.V., Ilinsky Y.Y., Rauschenbach I.Yu. The impact of FOXO on dopamine and octopamine metabolism in Drosophila under normal and heat stress conditions. Biol. Open. 2016; 5(11):1706-1711. DOI 10.1242/bio.022038.

14. Gruntenko N.E., Rauschenbach I.Yu. The role of insulin signalling in the endocrine stress response in Drosophila melanogaster: a minireview. Gen. Comp. Endocrinol. 2018;258:134-139. DOI 10.1016/j.ygcen.2017.05.019.

15. Guio L., Barron M.G., Gonzalez J. The transposable element BariJheh mediates oxidative stress response in Drosophila. Mol. Ecol. 2014;23:2020-2030.

16. Hwangbo D.S., Gershman B., Tu M.P., Palmer M., Tatar M. Drosophila dFOXO controls lifespan and regulates insulin signalling in brain and fat body. Nature. 2004;429(6991):562-566. DOI 10.1038/nature02549.

17. Ja W.W., Carvalho G.B., Mak E.M., de la Rosa N.N., Fang A.Y., Liong J.C., Brummel T., Benzer S. Prandiology of Drosophila and the CAFE assay. Proc. Natl. Acad. Sci. USA. 2007;104(20):8253-8256.10.1073/pnas.0702726104.

18. Jünger M.A., Rintelen F., Stocker H., Wasserman J.D., Végh M., Radimerski T., Greenberg M.E., Hafen E. The Drosophila forkhead transcription factor FOXO mediates the reduction in cell number associated with reduced insulin signaling. J. Biol. Chem. 2003;2(3):20. DOI 10.1186/1475-4924-2-20.

19. Kaluev A.V. Problems of Studying Stressful Behavior, Kiev: KSF Publ., 1999. (in Russian)

20. Koyama T., Texada M.J., Halberg K.A., Rewitz K. Metabolism and growth adaptation to environmental conditions in Drosophila. Cell. Mol. Life Sci. 2020;77:4523-4551.

21. Livak K.J., Schmittgen T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods. 2001;25(4):402-408. DOI 10.1006/meth.2001.1262.

22. Lubawy J., Urbanski A., Colinet H., Pfluger H.-J., Marciniak P. Role of the insect neuroendocrine system in the response to cold stress. Front. Physiol. 2020;11:376. DOI 10.3389/fphys.2020.00376.

23. Mattila J., Hietakangas V. Regulation of carbohydrate energy metabolism in Drosophila melanogaster. Genetics. 2017;207(4):1231-1253. DOI 10.1534/genetics.117.199885.

24. Miyashita A., Adamo S.A. Stayin’ alive: Endocrinological stress responses in insects. In: Saleuddin S., Lange A., Orchard I. (Eds.). Advances in Invertebrate Endocrinology. Toronto: Apple Acad. Press, 2020;283-325.

25. Murillo-Maldonado J.M., Sánchez-Chávez G., Salgado L.M., Salceda R., Riesgo-Escovar J.R. Drosophila insulin pathway mutants affect visual physiology and brain function besides growth, lipid, and carbohydrate metabolism. Diabetes. 2011;60(5):1632-1636. DOI 10.2337/db10-1288.

26. Okamoto N., Nakamori R., Murai T., Yamauchi Y., Masuda A., Nishimura T. A secreted decoy of InR antagonizes insulin/IGF signaling to restrict body growth in Drosophila. Genes Dev. 2013;27(1):87-97. DOI 10.1101/gad.204479.112.

27. Ponton F., Chapuis M.-P., Pernice M., Sword G.A., Simpson S.J. Evaluation of potential reference genes for reverse transcriptionqPCR studies of physiological responses in Drosophila melanogaster. J. Insect Physiol. 2011;57:840-850. DOI 10.1016/j.jinsphys.2011.03.014.

28. Puig O., Marr M.T., Ruhf M.L., Tjian R. Control of cell number by Drosophila FOXO: downstream and feedback regulation of the insulin receptor pathway. Genes Dev. 2003;17(16):2006-2020. DOI 10.1101/gad.1098703.

29. Rabasa C., Dickson S.L. Impact of stress on metabolism and energy balance. Curr. Opin. Behav. Sci. 2016;9:71-77. DOI 10.1016/j.cobeha.2016.01.011.

30. Rauschenbach I.Y., Karpova E.K., Burdina E.V., Adonyeva N.V., Bykov R.A., Ilinsky Y.Y., Menshanov P.N., Gruntenko N.E. Insulin-like peptide DILP6 regulates juvenile hormone and dopamine metabolism in Drosophila females. Gen. Comp. Endocrinol. 2017; 243:1-9. DOI 10.1016/j.ygcen.2016.11.004.

31. Slack C., Giannakou M.E., Foley A., Goss M., Partridge L. dFOXO-independent effects of reduced insulin-like signaling in Drosophila Aging Cell. 2011;10(5):735-748. DOI 10.1111/j.1474-9726.2011.00707.x.

32. Slaidina M., Delanoue R., Grönke S., Partridge L., Leopold P. A Drosophila insulin-like peptide promotes growth during nonfeeding states. Dev. Cell. 2009;17(6):874-884. DOI 10.1016/j.devcel.2009.10.009.

33. Van Handel E. Rapid determination of total lipids in mosquitoes. J. Am. Mosq. Control Assoc. 1985;1:302-304.

34. Williams M.J., Wang Yi., Klockars A., Lind P.M., Fredriksson R., Schiöth H.B. Exposure to bisphenol A affects lipid metabolism in Drosophila melanogaster. Basic Clin. Pharmacol. Toxicol. 2014; 114(5):414-420. DOI 10.1111/bcpt.12170.