Interaction between sleep and metabolism in Drosophila with altered octopamine signaling

Abstract

Sleep length and metabolic dysfunction are correlated, but the causal relationship between these processes is unclear. Octopamine promotes wakefulness in the fly by acting through the insulin-producing cells (IPCs) in the fly brain. To determine if insulin signaling mediates the effects of octopamine on sleep:wake behavior, we assayed flies in which insulin signaling activity was genetically altered. We found that increasing insulin signaling does not promote wake, nor does insulin appear to mediate the wake-promoting effects of octopamine. Octopamine also affects metabolism in invertebrate species, including, as we show here, Drosophila melanogaster. Triglycerides are decreased in mutants with compromised octopamine signaling and elevated in flies with increased activity of octopaminergic neurons. Interestingly, this effect is mediated at least partially by insulin, suggesting that effects of octopamine on metabolism are independent of its effects on sleep. We further investigated the relative contribution of metabolic and sleep phenotypes to the starvation response of flies with altered octopamine signaling. Hyperactivity (indicative of foraging) induced by starvation was elevated in octopamine receptor mutants, despite their high propensity for sleep, indicating that their metabolic state dictates their behavioral response under these conditions. Moreover, flies with increased octopamine signaling do not suppress sleep in response to starvation, even though they are normally hyper-aroused, most likely because of their high triglyceride levels. Together, these data suggest that observed correlations between sleep and metabolic phenotypes can result from shared molecular pathways rather than causality, and environmental conditions can lead to the dominance of one phenotype over the other.

FIGURE 1.

The insulin pathway does not mediate wake-promoting effects of octopamine. A, total sleep over a 24-hour period in female flies overexpressing ILP2 in ilp2-producing neurons (ilp2-Gal4<UAS-ilp2), compared with control flies that contain the ilp2-Gal4 or UAS-ilp2 transgene only. Total sleep is shown as mean ± S.E. n = 18 for each genotype. B, nighttime sleep in flies with increased octopamine signaling in the presence or absence of ilps 2 and 3 is shown as mean ± S.E. n≥12 for each genotype. *, p < 0.01 as compared with 21 °C baseline using Student's t test. C and D, total sleep in flies with altered insulin signaling. Either a fat body driver (yolk-Gal4) (C), or a pan-neuronal driver (Elav-Gal4 or an RU486 inducible ElavGS-Gal4) (D), was used to express UAS-dAkt RNAi, UAS-myrAKT or UAS-dInR^A1325D (constitutively active). ElavGS-Gal4 was activated by RU486 in 1% ethanol (EtOH) for 4 days and sleep in these flies was compared with that in flies given 1% EtOH without RU486. For other manipulations, flies containing both the Gal4 and UAS transgenes (Gal4<UAS) are compared with their Gal4 and UAS transgene alone controls. Total sleep is shown as mean ± S.E. n = 32 for each genotype. *, p < 0.01 as compared with each control line (Gal4 and UAS alone) using one-way ANOVA followed by Tukey-HSD post hoc test. ElavGS = Elav Gene Switch; myrAKT = myristylated AKT; dInR = Drosophila Insulin Receptor.

FIGURE 2.

Triglyceride levels are changed in flies with altered octopamine signaling. A, image shows a control fly (UAS-NaChBac/+) on the left and a fly with increased octopamine signaling, through activation of octopaminergic neurons (Tdc2-Gal4<UAS-NaChBac), on the right. Note the enlarged abdomen of the Tdc2-Gal4<UAS-NaChBac flies. B, octopamine signaling was increased either throughout development (Tdc2-Gal4<UAS-NaChBac) or conditionally during adulthood (Tdc2-Gal4<UAS-TrpA1) and triglyceride/total protein ratios were compared with those in control flies possessing only the Gal4 or UAS transgene. The effect of octopamine on triglycerides was reduced in an ilp2-3 mutant background. Octopamine signaling was decreased using an octopamine receptor mutant, oamb²⁸⁶, and triglyceride levels were compared with those of their background control (w1118). Each experiment was performed three times, and values represent mean ± S.E. of the pooled data. n ≥ 12 for each genotype. *, p < 0.05 relative to the control lines carrying Gal4 and UAS transgenes alone, as determined by one-way ANOVA followed by Tukey-HSD post hoc test. Ψ, p < 0.01 by Student's t test. Triglycerides were also analyzed using Thin Layer Chromatography in (C) oamb²⁸⁶ mutants, (D) flies with increased octopaminergic signaling (Tdc2-Gal4<UAS-TrpA1) on standard food and (E) flies with increased octopaminergic signaling (Tdc2-Gal4<UAS-NaChBac) on low nutrient food (2% agar, 5% sucrose). Arrows point to the butter standard. Each duplicate spot (indicated by a line below the two spots) on the plate represents a technical replicate for that genotype. Each experiment was replicated two or more times and a representative image is shown.

FIGURE 3.

Expression of enzymes controlling the synthesis and breakdown of lipids is altered in oamb mutants. Analysis of transcript levels of enzymes involved in either lipid synthesis (dFAS, dACC, dATPCL) or lipid breakdown (bmm, dCPTI) in the fat bodies of oamb mutants and their background control (w1118). The experiment was performed twice with the data pooled to include six biological replicates for each genotype. Values represent mean ± S.E. *, p <0.05 by Student's t test.

FIGURE 4.

Reduced sleep does not cause increased triglycerides. Triglyceride/total protein ratios of short sleeping mutants sleepless (sss) and fumin (fmn) were compared with their respective background controls. Triglycerides were also measured in flies where sleep was reduced in a wild type genetic background by expressing a constitutively active form of PKA (mc*) in the mushroom body using an RU486 inducible driver (MBGS). Controls were given 1% ethanol (EtOH) without RU486. Plotted values equal the average of data pooled from at least three experiments ± S.E. n ≥ 13 for each genotype *, p < 0.01 by Student's t test compared with each appropriate control.

FIGURE 5.

The response to starvation is dictated by metabolic need, rather than sleep pressure. A, lifespan of flies with increased (Tdc2-Gal4<UAS-NaChBac) or decreased (oamb²⁸⁶) octopamine signaling on starvation medium. Survival time reflects lifespan following the start of food deprivation. The number of dead flies was determined every 3 h. The graph summarizes the results of at least two experiments. B, oamb²⁸⁶ mutants have exaggerated activity in response to starvation although they reduce sleep similar to controls. Total sleep over a 24-hour period was determined for the first day flies were starved on 2% agar (starvation) and the day before starvation in which flies were given sucrose food (baseline). C, flies with increased octopamine signaling do not suppress sleep in response to starvation. Flies expressing NaChBac in octopaminergic cells also display increased activity in response to starvation; however, they do not display similar reductions in sleep. Activity is plotted as the average of data pooled from three independent experiments. The activity value equals the average number of beam crossings over a period of time (1, 3, 6, 12, 24 h) following starvation. Error bars reflect ± S.E. Statistical significance was determined using pooled data. Ψ, p < 0.01 and *, p < 0.05 correspond to comparisons of activity and sleep following starvation to the baseline values for each genotype by one-way ANOVA followed by Tukey-HSD posthoc test and Student's t test, respectively.