Sleep-Dependent Modulation of Metabolic Rate in Drosophila

Abstract

Study objectives: Dysregulation of sleep is associated with metabolic diseases, and metabolic rate (MR) is acutely regulated by sleep-wake behavior. In humans and rodent models, sleep loss is associated with obesity, reduced metabolic rate, and negative energy balance, yet little is known about the neural mechanisms governing interactions between sleep and metabolism.

Methods: We have developed a system to simultaneously measure sleep and MR in individual Drosophila, allowing for interrogation of neural systems governing interactions between sleep and metabolic rate.

Results: Like mammals, MR in flies is reduced during sleep and increased during sleep deprivation suggesting sleep-dependent regulation of MR is conserved across phyla. The reduction of MR during sleep is not simply a consequence of inactivity because MR is reduced ~30 minutes following the onset of sleep, raising the possibility that CO2 production provides a metric to distinguish different sleep states in the fruit fly. To examine the relationship between sleep and metabolism, we determined basal and sleep-dependent changes in MR is reduced in starved flies, suggesting that starvation inhibits normal sleep-associated effects on metabolic rate. Further, translin mutant flies that fail to suppress sleep during starvation demonstrate a lower basal metabolic rate, but this rate was further reduced in response to starvation, revealing that regulation of starvation-induced changes in MR and sleep duration are genetically distinct.

Conclusions: Therefore, this system provides the unique ability to simultaneously measure sleep and oxidative metabolism, providing novel insight into the physiological changes associated with sleep and wakefulness in the fruit fly.

Keywords: Drosophila; calorimetry; metabolism; respirometry; sleep.

Figure 1—

A system to measure MR in single flies. (A) MR was measured through indirect calorimetry. A stop-flow respirometry system measured the CO₂ produced by single flies placed inside of a 70 mm long × 20 mm diameter glass tube. Each fly had access to 1% agar and 5% sucrose. Activity and sleep were measured simultaneously as MR using a Drosophila Locomotor Activity Monitor with three infrared beams running through each behavior chamber. The computer counted the number of beam breaks. (B) A representative 5-minute reading, with the activity in number of beam crosses and the amount of CO₂ produced by each fly over time. (C) The MR and activity for one female fly. (D) The MR and activity for one male fly. (E) The activity of female (N = 24; p < .0001) and male (N = 35; p < .0001) flies in beam crosses per hour, over 12 hours of day and night (two-way ANOVA F_(1,114) = 171.9, p < .0001). Condition-by-sex interaction is significant (two-way ANOVA F_(1,114) = 15.30, p < .001). (F) The MR of female (N = 24; p < .01) and male (N = 35; p < .01) flies as CO₂ produced per hour, over 12 hours of day and night (two-way ANOVA F_(1,114) = 21.27, p < .0001). Condition-by-sex interaction is not significant (two-way ANOVA F_(1,114) = 0.4137, p > .50). (G) Linear regression of absolute vCO₂ readout versus activity of female flies (N = 24 each bin; R² = 0.120) and (H) male flies (N = 35 each bin; R² = 0.064). Gray dashed lines indicate 95% confidence interval. One-way ANOVA comparing the vCO₂ at the 1–10 beam crossings bin to each subsequent beam crossing bin: females >60 crossings (N = 24 each bin; p < .05) and males >60 crossings (N = 35 each bin; p < .05). ANOVA = analysis of variance; IR = infrared.

Figure 2—

MR is reduced during sleep state. Female control flies (w¹¹¹⁸) were allowed to acclimate in the system for 24 hours. (A) MR shows an inverse pattern to their sleep (N = 24). (B) Total minutes of sleep per 12 hours of day and night for B (N = 24; p < .001). (C) The MR of female flies as CO₂ produced per hour, over 24 hours of day and night (N = 24; p < .002). (D) The MR throughout a single, representative sleep bout during the night. (E) Linear regression model comparing percent change in MR versus sleep duration, binned per 5 minutes (N = 24; R² = 0.266). Gray dashed lines indicate 95% confidence interval. One-way ANOVA comparing the initial percent change in MR at the 10-minute sleep bin to each subsequent sleep bin reveals significant differences after 35 minutes asleep (N = 24 each sleep bin; p < .05). ANOVA = analysis of variance.

Figure 3—

MR is elevated during sleep deprivation and reduced during rebound. (A) Female control flies (w¹¹¹⁸) were acclimated during the day (ZT0-12). Mechanical sleep deprivation was applied during the 12-hour night (ZT12-24), and recovery was assessed the following day (ZT0-12). (B) Sleep-deprived flies (N = 15; purple) sleep more during the first 6 hours of daytime following deprivation (ZT0-6) relative to undisturbed controls (N = 15; black). (C) Quantification of total sleep shows that flies were sufficiently sleep deprived during nighttime (ZT18-24; p < .0001) and demonstrated increased sleep during the recovery period (ZT0-6; p < .0001). (D) Hourly profile of MR in sleep deprived and control flies. (E) Quantification MRs demonstrates elevated MR during sleep deprivation (ZT18-24; p < .0001) and reduced MR during recovery (ZT0-6; p < .0001). MR during recovery in sleep-deprived flies is comparable to levels of control flies during normal nighttime sleep (p > .327). (F) Regression analysis comparing percent change in MR versus sleep duration, binned per 5 minutes (N = 15; R² = 0.174). Gray dashed lines indicate 95% confidence interval. One-way ANOVA comparing the initial percent change in MR at 10-minute sleep bin to each subsequent sleep bin reveals significant differences after 35 minutes asleep (N = 15 each sleep bin; p < .05). ANOVA = analysis of variance.

Figure 4—

Reduced MR during pharmacologically induced sleep. (A) Female w¹¹¹⁸ flies were loaded on sucrose or sucrose containing 0.1 mg/mL gaboxadol 2 hours before lights out (ZT10), acclimated to the system for 12 hours during the night phase and were measured for 12 hours (ZT0-12) during the following day. (B) Daytime sleep was significantly elevated in gaboxadol-treated flies (green) compared to flies fed sucrose alone (black). (C) Quantification of total sleep reveals gaboxadol-treated flies (N = 15) sleep significantly longer than untreated controls (N = 14; p < .0001). (D) MR was reduced throughout the 12-hour day. (E) Quantification of mean MR reveals a significant reduction in gaboxadol-treated flies (N = 15) compared to controls (N = 14; p < .0001). (F) Linear regression of percent change in MR versus sleep duration, binned per 5 minutes (N = 15; R² = 0.200). Gray dashed lines indicate 95% confidence interval. One-way ANOVA comparing the initial percent change in MR at the 10-minute sleep bin to each subsequent sleep bin reveals significant differences after 30 minutes asleep. (N = 15 each sleep bin; p < .05). ANOVA = analysis of variance.

Figure 5—

MR and sleep are reduced in starved flies. (A) Flies were fed or starved while acclimating to the system for 12 hours during the day (ZT0-12) before measurement throughout the night (ZT12-24). (B) Flies starved on agar (blue) slept less than flies housed on 5% sucrose (black) during the 12-hour night period. (C) Quantification of total sleep over the 12-hour night period reveals a significant reduction in starved flies (N = 30) compared to fed controls (N = 29; p < .0001). (D) MR is lower throughout the 12-hour nighttime period in starved flies. (E) Quantification of mean vCO₂ production over this period reveals a signification reduction in starved animals (N = 30) relative to controls (N = 29; p < .01). (F) Regression analysis comparing percent change in MR versus sleep duration, binned per 5 minutes reveals a correlation in fed flies (N = 29; R² = 0.201), but little effect in starved flies (N = 26 each sleep bin, four flies did not have any sleep bouts when starved; R² = 0.067). Gray dashed lines indicate 95% confidence interval of each line. Comparison of the regression lines indicate that the slopes are different between the fed versus starved state (F = 5.319; p < .05). One-way ANOVA comparing the initial percent change in MR at the 10-minute sleep bin to each subsequent sleep bin within each group reveals significant differences after 40 minutes asleep in fed flies (N = 29 each sleep bin; p < .05) and no significant differences in starved flies (N = 26 each sleep bin). ANOVA = analysis of variance.

Figure 6—

Sleep-dependent changes in metabolism are intact in trsn^null flies. (A) Sleep did not significantly differ between trsn^null flies housed on sucrose or starved on agar alone. (B) Quantification of total nighttime sleep (ZT12-ZT24) revealed sleep is significantly lower in w¹¹¹⁸ control flies (N = 30) housed on agar compared to fed (N = 28; p < .0001), while there is no significant difference between trsn^null flies (N = 28) housed on 5% sucrose or agar alone (N = 25; p > .05; two-way ANOVA F_(1,107) = 42.52, p < .0001). Treatment-by-genotype interaction is significant (two-way ANOVA F_(1,107) = 11.24), p < .01). (C) MR is lower in control and trsn^null flies housed on agar compared to flies housed on 5% sucrose. (D) Quantification revealed MR is lower in both starved trsn^null flies and controls (w¹¹¹⁸, p < .0001; trsn^null, p < .01; two-way ANOVA F_(1,107) = 28.22, p < .0001). There is no effect of treatment-by-genotype interaction (two-way ANOVA F_(1,107) = 0.7162, p > .30). (E) Applied linear regression model comparing percent change in MR versus sleep duration, binned per 5 minutes reveals a correlation in w¹¹¹⁸ fed flies (N = 28; R² = 0.208), but only a weak effect in w¹¹¹⁸ starved flies (N = 25, 5 flies did not sleep on agar; R² = 0.097). Gray dashed lines indicate 95% confidence interval of each line. Comparison of the regression lines indicate that the slopes are different between the w¹¹¹⁸ fed versus starved state (F = 7.09725, p< .01). One-way ANOVA comparing the initial percent change in MR at the 10-minute sleep bin to each subsequent sleep bin within each group reveals significant differences after 40 minutes asleep in fed flies (N = 28 each sleep bin; p < .05) and differences in starved flies beyond 55 minutes (N = 25 each sleep bin; p < .05). (F) Regression analysis model comparing percent change in MR versus sleep duration, binned per 5 minutes reveals a correlation in trsn^null fed flies (N = 28; R² = 0.201), but only a weak effect in trsn^null starved flies (N = 25; R² = 0.183). Gray dashed lines indicate 95% confidence interval of each line. Comparison of the regression lines indicates that the slopes do not differ between the trsn^null fed versus starved state (F = 5.0557, p < .05). One-way ANOVA comparing the initial percent change in MR at the 10-minute sleep bin to each subsequent sleep bin within each group reveals significant differences after 25 minutes asleep in trsn^null fed flies (N = 28 each sleep bin; p < .05) and differences in trsn^null starved flies beyond 30 minutes (N = 25 each sleep bin; p < .05). ANOVA = analysis of variance.