Serine metabolism in the brain regulates starvation-induced sleep suppression in Drosophila melanogaster

Abstract

Sleep and metabolism are physiologically and behaviorally intertwined; however, the molecular basis for their interaction remains poorly understood. Here, we identified a serine metabolic pathway as a key mediator for starvation-induced sleep suppression. Transcriptome analyses revealed that enzymes involved in serine biosynthesis were induced upon starvation in Drosophila melanogaster brains. Genetic mutants of astray (aay), a fly homolog of the rate-limiting phosphoserine phosphatase in serine biosynthesis, displayed reduced starvation-induced sleep suppression. In contrast, a hypomorphic mutation in a serine/threonine-metabolizing enzyme, serine/threonine dehydratase (stdh), exaggerated starvation-induced sleep suppression. Analyses of double mutants indicated that aay and stdh act on the same genetic pathway to titrate serine levels in the head as well as to adjust starvation-induced sleep behaviors. RNA interference-mediated depletion of aay expression in neurons, using cholinergic Gal4 drivers, phenocopied aay mutants, while a nicotinic acetylcholine receptor antagonist selectively rescued the exaggerated starvation-induced sleep suppression in stdh mutants. Taken together, these data demonstrate that neural serine metabolism controls sleep during starvation, possibly via cholinergic signaling. We propose that animals have evolved a sleep-regulatory mechanism that reprograms amino acid metabolism for adaptive sleep behaviors in response to metabolic needs.

Keywords: serine; sleep regulation; starvation.

Fig. 1.

Transcriptome analyses of starved Drosophila brains identify up-regulation of serine biosynthesis pathway. (A) Schematic diagram depicting RNA-seq experimental design. Wild-type flies were fed 5% sucrose/1% agar (Ctrl) or deprived of sucrose for 6 and 24 h (FD6 and FD24). (B) Scatter plots demonstrate log counts per million (cpm) vs. log fold-change (FC) in expression of brains starved for 6 and 24 h. Blue dots represent DEGs (FDR < 0.05). (C) Venn diagram showing the number of genes that are regulated during short-term (FD6) and long-term (FD24) starvation. (D) Gene ontology analysis of genes that are up-regulated exclusively in FD6 and FD24. (E) Schematic diagram of the serine metabolic pathway. (F) Heat-map of expression level of genes involved in the serine metabolic pathway in fed (Ctrl FD6 and Ctrl FD24) and starved conditions (FD6 and FD24). Colors indicate the log2 values of normalized read counts.

Fig. 2.

Starvation-induced aay expression elevates free serine levels in heads and supports sleep suppression during starvation. (A) Schematic diagram illustrating KG05974 insertion near the 5′UTR of aay locus. (B) Western blot of head extracts in iso31 and aay^KG flies during fed and starved conditions. (C) Quantification of Western blots (n = 3) [F_(1,8) = 12.92; P = 0.0070]. (D) Serine concentrations in head extracts of iso31 control and aay^KG flies during fed and starved conditions (n = 3) [F_(1,8) = 14.53; P = 0.0052]. (E) Average sleep traces of iso31 control (black; n = 88), aay^KG/+ (light orange; n = 80), and aay^KG (orange; n = 78) flies. (F) Percentage sleep change during starvation in iso31 and aay^KG/+ controls vs. aay^KG flies. ns, P > 0.05; *P < 0.05; **P < 0.005; ***P < 0.0005; ****P < 0.0001. All error bars represent SEM.

Fig. 3.

stdh metabolizes free serine and limits starvation-induced sleep suppression. (A) Schematic diagram illustrating C04459 insertion in the intron of the stdh locus. (B) stdh transcript levels are strongly repressed in stdh^pBac flies (n = 3). (C) Serine and (D) threonine concentrations in head extracts of iso31 control and stdh^pBac flies during fed and starved conditions (n = 3). (E) Average sleep traces of iso31 (black; n = 91), stdh^pBac/+ (light green; n = 81), and stdh^pBac flies (green; n = 83). (F) Percentage sleep change during starvation in iso31 and stdh^pBac/+ controls vs. stdh^pBac flies. ns, P > 0.05; *P < 0.05; **P < 0.005; ****P < 0.0001. All error bars represent SEM.

Fig. 4.

aay and stdh act in the same genetic pathway to titrate free serine levels and control sleep during starvation. (A) Average sleep traces of iso31 (black; n = 116), aay^KG (orange; n = 81), stdh^pBac (green; n = 104), and double mutants (purple; n = 160). (B) aay^KG masks the exaggerated starvation-induced sleep suppression of stdh^pBac flies [F_(1,457) = 6.035; P = 0.0144]. (C) Comparison of serine, glycine, and threonine levels in head extracts of iso31 and mutants in starved condition (n = 3). ns, P > 0.05; *P < 0.05; **P < 0.005; ***P < 0.0005; ****P < 0.0001. All error bars represent SEM.

Fig. 5.

Sleep regulation by the serine metabolic pathway implicates cholinergic signaling. (A) Percentage sleep change during starvation in flies with pan-neuronal and glial knockdown of aay transcripts (aay RNAi #1/+, n = 37; elav-Gal4/+, n = 46; elav-Gal4 > aay RNAi #1, n = 42; nrv2-Gal4/+, n = 29; nrv2-Gal4 > aay RNAi #1, n = 21). (B) Percentage sleep change during starvation when aay transcripts are depleted in mushroom body (MB), fan-shaped body (FB), pars intercerebralis (PI), leucokinin, clock, and various neurotransmitter-related regions in the brain (n = 7–33). (C) Dose-dependent effect of MCA administration on stdh^pBac flies during starvation. The 200 μM MCA effect: [F_(1,281) = 3.637; P = 0.0575] and 800 μM MCA effect: [F_(1,284) = 6.071; P = 0.0143]. iso31 0 μM, n = 71; iso31 200 μM, n = 79; iso31 800 μM, n = 81; stdh^pBac 0 μM, n = 66; stdh^pBac 200 μM, n = 69; and stdh^pBac 800 μM, n = 70. ns, P > 0.05; *P < 0.05; **P < 0.005; ***P < 0.0005; ****P < 0.0001. All error bars represent SEM.