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.

Statement of Significance

Metabolic disorders are associated with sleep disturbances, yet our understanding of the mechanisms underlying interactions between sleep and metabolism remains limited. Here, we describe a novel system to simultaneously record sleep and metabolic rate in single Drosophila. Our findings reveal that uninterrupted sleep bouts of 30 minutes or greater are associated with a reduction in metabolic rate providing a physiological readout of sleep. Use of this system, combined with existing genetic tools in Drosophila, will facilitate identification of novel sleep genes and neurons, with implications for understanding the relationship between sleep loss and metabolic disease.

INTRODUCTION

Dysregulation of sleep is strongly linked to metabolism-related pathologies, and reciprocal interactions between sleep and metabolism suggest these processes are integrated at the cellular and molecular levels.^1,2 In mammals, metabolic rate (MR) is reduced during sleep raising the possibility that sleep provides a mechanism of energy conservation or partitioning.³ Although a reduction in MR and energy expenditure during sleep has been documented in mammalian and avian species,^4,5 little is known about the genetic and neural mechanisms governing the effects of sleep on MR. The fruit fly, Drosophila melanogaster, displays all the behavioral characteristics of sleep and provides a powerful system for genetic investigation of interactions between sleep and diverse physiological processes.^2,6,7 Here, we describe a novel single-fly respirometry assay in the fruit fly, designed to simultaneously measure sleep and whole-body MR that allows for genetic interrogation of the mechanisms regulating interactions between these processes.

Sleep is characterized by physiological changes in brain activity or through the behavioral correlates that accompany these changes.⁸ Flies, like mammals, display distinct electrophysiological patterns that correlate with wake and rest.^9,10 Additionally, flies display all the behavioral hallmarks of sleep including extended periods of behavioral quiescence, rebound following deprivation, increased arousal threshold, and species-specific posture.^6,7 Sleep in Drosophila is typically defined by 5 minutes of behavioral quiescence because this correlates with other behavioral characteristics used to define sleep.⁷ Although these behavioral metrics of sleep have been studied extensively, significantly less is known about physiological changes associated with sleep in flies.

In rodents and humans, MR is elevated in response to sleep deprivation and reduced during sleep, supporting the notion that metabolic processes are acutely regulated by sleep state.^11–13 In flies and other small insects, stop-flow respirometry can be used to monitor CO₂ production, a by-product of oxidative metabolism and a proxy for MR.¹⁴ Here, we describe a system to simultaneously measure sleep and MR in individual fruit flies. Our findings reveal that MR is reduced when flies sleep, and uninterrupted sleep bouts of ~30 minutes or greater are associated with an additional reduction in MR, indicating that flies exhibit sleep stages that are physiologically distinct. Further, we find that starvation inhibits sleep-associated reductions in MR, suggesting feeding state influence physiological changes associated with sleep. These findings suggest that sleep-dependent reductions in MR previously observed in mammals are conserved in the fruit fly and further support the notion that sleep provides a mechanism for energy conservation.

MethodS

Drosophila Maintenance and Fly Stocks

Flies were grown and maintained on standard food (Bloomington Recipe, Genesee Scientific). Flies were maintained in incubators (Powers Scientific; Dros52) at 25°C on a 12:12 light/dark cycle, with humidity set to 55%–65%. The wild-type line used in this manuscript is the w¹¹¹⁸ fly strain (Bloomington Stock #5905). The trsn^null allele is an excision of the trsn^EY06981 locus derived from mobilizing the EPgy2 insertion.¹⁵ This allele removes the entire coding region of the gene and represents a null mutation that has been outcrossed to the w¹¹¹⁸ background and has previously been described as Δtrsn.¹⁵ Unless noted in the figures, all experiments are performed in 3- to 5-day-old mated female flies.

Measurement of MR and Locomotor Activity

MR was measured at 25°C through indirect calorimetry, measuring the CO₂ production of individual flies with a Li-7000 CO₂ analyzer (LI-COR), which was calibrated with pure CO₂ before each run. A stop-flow, push-through respirometry setup was constructed using Sable Systems equipment (Sable Systems International). The experimental setup included sampling CO₂ from an empty chamber to assess baseline levels, alongside five behavioral chambers, each measuring CO₂ production of a single fly. The weight of flies used for analysis were not taken into account because body size and energy stores are not perturbed in trsn^null flies and do not vary significantly in w¹¹¹⁸ flies. Further, previous work using a comparable system suggests weight will have little effect on CO₂ measurements unless there is an excess of >50% differences in size between individuals.^14,16 To measure CO₂ output, air was flushed from each chamber for 50 seconds providing a readout of CO₂ accumulation over a 5-minute period. This 5-minute interval allows the coordinate and simultaneous activity-based assessment of sleep. The first 20 minutes of recordings were not included in analyses because this time was necessary to purge the system of ambient air and residual CO₂ from the closed system. Dehumidified, CO₂ free air was pumped through a mass flow control valve (Side-Trak 840 Series; Sierra Instruments, Inc.) to maintain the experimental flow rate of 100 mL/min. The air was then passed through water-permeable Nafion tubing (Perma Pure, LLC, Lakewood, New Jersey, USA, #TT-070) immersed in a reservoir containing deionized H₂O to rehumidify the air before reaching the behavioral chambers. Nonpermeable Bev-A line tubing (United States Plastic Corp., Lima, Ohio, USA, #56280) was used throughout the rest of the system.

Experiments were conducted by placing single flies in 70 mm × 20 mm glass tubes that fit a custom-built Drosophila Locomotor Activity Monitor (Trikinetics, Waltham, Massachusetts) with three sets of infrared (IR) beams for activity detection. The monitor was connected to a computer to record beam breaks every minute for each animal using standard Drosophila Activity Monitor (DAMS) activity software (Trikinetics, Waltham, Massachusetts) as previously described.¹⁷ These data were used to calculate sleep information by extracting immobility bouts of 5 minutes using a custom-generated python program. The total activity from all three beams was summed for each time point in order to determine overall activity. Video recordings for analysis of feeding activity were acquired using a handheld USB Digital microscope (Vivida, 2MP #eheV1-USBpro) camera at 12 fps with VirtualDub software (v.1.10.4). Each 60-minute video recording occurred between ZT01-04 to prevent circadian differences in sleep, feeding, and MR. During video recording, flies were simultaneously assayed for activity and MR, with the stop flow set to collect CO₂ output every 2 minutes. Videos were manually scored for feeding activity in corresponding 2-minute intervals as a “feeding” or “nonfeeding” bin.

Flies were briefly anesthetized using CO₂ for sorting at least 24 hours before the start of an experiment to allow for metabolic recovery. For all experiments, flies were loaded into chambers by mouth pipette to avoid confounding effects of anesthesia and allowed to acclimate in the system with the air flowing for 12–24 hours before behavior experiments, unless otherwise specified. To control for effects of diet composition, all experimental flies were fed a consistent diet. Each chamber contained a single food vial containing 1% agar plus 5% sucrose (Sigma) with red food coloring (McCormick), which we have previously shown to result in sleep comparable to standard fly food.¹⁸ For starvation experiments, flies had access to 1% agar dissolved in dH₂O and were acclimated for 12 hours during lights on with access to agar alone, with analyses beginning at ZT12 at lights off. All experimental runs included analysis of both experimental flies and relevant controls in a randomized order to account for any subtle variation between runs.

Pharmacology

Pharmacological-induced sleep was achieved through administration of gabaxodol (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol hydrochloride, THIP hydrochloride; Sigma Aldrich #85118-33-8) at the dosage of 0.1 mg/mL, as previously described.¹⁹ Gaboxadol was dissolved in dH₂O with 1% agar and 5% sucrose. Flies were loaded into the respirometry system with the gaboxadol 2 hours before lights off (ZT10) and were maintained on the drug throughout the duration of the experiment, as described in the text.

Sleep Deprivation

Flies were acclimated to the respirometry system during the daytime (ZT0-12). For mechanical sleep deprivation, flies were shaken every 2–3 minutes for 12 hours in the modified DAMs monitor/respirometry system throughout the nighttime (ZT12-24) while simultaneously measuring MR. The mechanical stimulus was applied using a vortexer (Fisher Scientific, MultiTube Vortexer) and a repeat cycle relay switch (Macromatic, TR63122). Sleep rebound and corresponding MR was measured the following day from ZT0-ZT12.

Sleep, Metabolic, and Statistical Analyses

Respirometry recordings were analyzed using ExpeData PRO software (Sable Systems International, v1.8.4). The CO₂ lag time from the chamber to the analyzer was corrected, the baseline was subtracted from each behavioral chamber, and the absolute CO₂ levels (ppm) was converted to μL/hr using the recorded air flow rate. Integrating the CO₂ trace revealed the total CO₂ produced, or the average MR, per fly for each recording. These data were exported to Excel, where metabolic output was matched to activity, and sleep analyses were performed using a custom python program. Since individual flies were measured for either a 12-hour or 24-hour experimental duration (described in text), our raw data included resampling of MR or beam crosses for each hour. To account for these repeated hourly measures, we determined the mean of the hourly readings for each individual fly before our statistical analyses represented in the graphs, meaning that each fly is represented once and the “N” reported in each figure specifically refers to the number of individual flies assayed in the experiment. To detect significant differences for activity (number of beam crosses), mean VCO₂ (μL/hour) or total sleep (minutes), we employed a Student t-test (day vs. night; untreated control vs. gaboxadol-treated; fed vs. starved), two-way analysis of variance (ANOVA) with Sidak’s multiple comparison correction (female, day vs. night; male, day vs. night), and a two-way ANOVA with Sidak’s multiple comparison correction (w¹¹¹⁸, fed vs. starved; trsn, fed vs. starved), when appropriate using InStat software (GraphPad Software 6.0). The two-tailed p-value used to test significance is denoted as p < .05.

To account for individual-specific differences in MR, we surveyed the MR throughout longer sleep bouts by calculating percent change in MR. This was determined by subtracting the MR during the first 5 minutes asleep from the MR during each of the subsequent 5 minutes asleep for the entire length of the sleep bout, divided by the MR during the first 5 minutes asleep, multiplied by 100 (eg, [{first 5 minutes MR} – {20 minutes MR}/{first 5 minutes MR}] × 100). We note some flies exhibited longer sleep bouts; however, this analysis was restricted to sleep bouts up to 60 minutes due to limited replicates with extended bout lengths. Moreover, a similar approach was employed as described above for analysis of MR for flies with repeated sleep bouts. If a single fly demonstrated multiple distinct sleep bouts, we determined the mean percent change in MR for each fly at each sleep bin. For these analyses, we performed a one-way ANOVA with Sidak correction comparing the initial percent change in MR (5-minute bin) to each of the subsequent sleep bins (15–60 minute bins at 5-minute intervals) using InStat software (GraphPad Software 6.0) with significance denoted as p < .05.

We applied a linear regression model to characterize the relationship between both absolute vCO₂ versus activity (number of beam crossings) and percent change in MR and sleep duration using InStat software (GraphPad Software 6.0) with significance denoted as p < .05. Comparison of slopes derived from regression lines in fed versus starved states was performed using analysis of covariance (F-statistic; GraphPad Software 6.0). Before modeling, we performed pretests, including: generation of residual versus fitted plots to determine homogeneity of variance, normal Q-Q plot, Pearson correlation table and linear model assumptions (B.L.U.E.). The culmination of these tests indicated that our data were both normally distributed and appropriate for linear regression modeling.

RESULTS

Long-Term Recordings of Sleep and MR

To simultaneously measure the effects of sleep on MR, we designed a stop-flow respirometry system coupled to a custom-built DAM system (Figure 1A). Each DAM chamber contained three IR beams for precise detection of locomotor activity of a single fly.²⁰ Humidified, fully oxygenated air was passed through each chamber, preventing desiccation and allowing for long-term recordings. After exiting the chamber, air was dehumidified and passed through a CO₂ analyzer. The system was set to a stop-flow configuration, where the CO₂ accumulation in each chamber was measured every 5 minutes, and these were matched to the corresponding locomotor activity of individual flies within this period (Figure 1B). Flies are diurnal with elevated locomotor levels during the day compared to night, and these activity patterns were maintained in the respirometry system in both male and female flies (Figure 1C and D), indicating that the moderate airflow used in this system does not disrupt sleep-wake behavior. In both male and female flies, the mean MR was elevated during the daytime compared to the night, supporting the notion that CO₂ production is associated with periods of high activity (Figure 1E and F). Examination of CO₂ levels in individual flies revealed a weak correlation in both females and males between total locomotor activity and CO₂ levels (Figure 1G and H). However, vCO₂ was significantly elevated in females with activity of >60 beam breaks and males >50 beam breaks per 5-minute bin compared to the 1–10 beam breaks bin, suggesting MR is elevated during periods of robust activity (Figure 1G and H). Therefore, this system effectively measures locomotor activity and MR simultaneously in individual Drosophila.

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.

MR Is Reduced in Sleeping Drosophila

Five minutes of immobility in Drosophila associates with relevant behavioral and physiological sleep metrics, allowing for sleep duration to be inferred from periods of behavioral quiescence.^7,10 To measure MR during sleep, female flies were acclimated in the locomotor chambers for 24 hours, followed by continuous measurements of sleep and MR for an additional 24 hours. Flies slept significantly more during the night (ZT12-24), which corresponded with a reduction in MR (Figure 2A–C). To determine whether changes in MR are associated with sleep bout duration, we investigated changes in CO₂ production during sleep bouts. CO₂ production during a single representative 60-minute sleep bout revealed a reduction in MR as sleep progressed (Figure 2D). To account for individual variation between replicates, change in MR was calculated as percent change for each 5-minute interval throughout the sleep bout compared to the first 5 minutes of sleep. To avoid confounds resulting from circadian differences in MR, analysis was limited to nighttime sleep. Regression analysis revealed a significant relationship between of vCO₂ and sleep bout length (Figure 2E). Comparing the average percent change in MR during sleep for each individual bout revealed MR was significantly reduced following 35 minutes of sleep, indicating that longer periods of uninterrupted sleep are associated with reduced MR. Percent change in MR continued to decline as sleep progressed until reaching a maximum percent change in MR of ~−12% to 15% after 50 minutes of sleep. To confirm that reduction of MR during sleep is not simply due to lack of feeding activity, we compared MR during feeding and nonfeeding bins from ZT1-ZT3 and did not detect significant differences in MR between feeding and waking nonfeeding periods (Supplementary Figure S1). Moreover, we performed standard allometric analysis of body size versus MR to identify if weight variation among individual flies could function as a covariate affecting MR^21,22 and determined that there is no effect of variation in body weight on MR (n = 34, R² = 0.030). Therefore, reduced CO₂ production is associated with consolidated sleep bouts, revealing that MR can be functionally separated from overall activity.

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.

MR During Sleep Deprivation and Rebound Sleep

To further examine the relationship between sleep and MR, we sleep deprived flies during the night (ZT12-24) and measured vCO₂ during deprivation and recovery (Figure 3A). Consistent with previous findings, sleep deprivation significantly increased sleep the following day (ZT0-6) compared to nonsleep deprived controls (Figure 3B–C). MR was elevated in sleep-deprived flies during deprivation (ZT12-24), and reduced during recovery (ZT0-6), fortifying the notion that reduced MR is associated with sleep. There was a significant correlation between MR and sleep bout duration, indicating that similar to nighttime sleep in undisturbed flies, prolonged bouts of daytime sleep are associated with reduced MR (Figure 3F). Rebound sleep demonstrated a significant reduction in metabolic as sleep duration progressed beyond 35 minutes (Figure 3F), further supporting the notion that daytime rebound recapitulates physiologically similar sleep-associated metabolic changes to nighttime sleep.

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.

MR Is Reduced During Pharmacologically Induced Sleep

Gamma-amino butyric acid (GABA) signaling promotes sleep in diverse species, and the GABA-A receptor agonist gabaxodol potently induces sleep in Drosophila.^19,23–25 To determine the effects of pharmacologically induced sleep on MR, we housed flies on agar containing 0.1 mg/mL gaboxadol and 5% sucrose in the respirometry chambers and measured the effects on sleep and MR (Figure 4A). Consistent with previous studies, sleep was elevated in gaboxadol-treated flies compared to controls throughout the 12-hour daytime recording¹⁹ (Figure 4B and C). Notably, MR was reduced in gaboxadol-treated flies during the daytime compared to controls, confirming that pharmacologically induced sleep lowers MR (Figure 4D and E). These experiments were limited to analysis of daytime sleep, therefore, we could not determine percent change in MR of untreated w¹¹¹⁸ flies across sleep bouts, since control flies sleep very little during the day in this paradigm. Sleep bout length in gaboxadol-treated flies was associated with reduced MR (Figure 4F). Moreover, comparison of the of percent change in MR of each subsequent sleep bin relative to the first change at 10 minutes shows a robust reduction in MR in gaboxadol-treated flies after 30 minutes of sleep (Figure 4F). Because the percent change in MR is comparable to the MRs of wild-type flies during night sleep, it is possible that pharmacologically induced daytime sleep is physiologically comparable to nighttime sleep.

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.

The Effects of Starvation on Sleep and MR

In mammals, starvation potently suppresses sleep and MR.²⁶ Further, flies suppress sleep shortly after the onset of food deprivation, presumably to increase foraging behavior.^18,27 To determine how starvation-induced sleep suppression impacts metabolic function, we compared the MR of fed and starved female w¹¹¹⁸ flies. Flies were acclimated for 12 hours on food or agar, and MR was measured during the 12-hour night phase (ZT12-24; Figure 5A). In agreement with previous findings, sleep was reduced in starved flies throughout the 12-hour recording period^28,29 (Figure 5B and C). Despite the loss of sleep in starved flies, MR was lower in starved animals compared to fed counterparts, providing further support that MR in Drosophila is modulated independently from locomotor activity (Figure 5D and E). There was a significantly stronger relationship between sleep bout length and MR in fed flies, providing evidence that starvation impairs sleep-associated physiological changes on MR (Figure 5F). To determine the effect of starvation on sleep-dependent regulation of MR, we compared the MR of each sleep bout in fed and starved animals. In fed flies, MR was reduced following 40 minutes of sleep compared to the first 5 minutes of sleep, yet when starved, MR is not significantly reduced as sleep progresses, further supporting the notion that starvation impedes sleep (Figure 5F). Taken together, these findings reveal that CO₂ production is reduced in starved flies without affecting sleep-dependent changes in MR.

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.

Metabolic Changes During Sleep Are Intact in translin Mutant Flies

It is possible that shared genes regulate starvation-induced reductions in sleep duration and sleep-dependent regulation of MR. We previously identified the RNA-binding protein translin (trsn), as essential for starvation-induced sleep suppression.²⁹ Energy stores and feeding behavior are normal in trsn deficient flies, yet they fail to suppress sleep in response to starvation, suggesting trsn is required for the integration of sleep and metabolic state.²⁹ To determine whether trsn affects MR, we measured sleep and MR in fed and starved trsn mutant flies. Flies were loaded into the respirometry system and allowed to acclimate for 12 hours during the day. Sleep and MR were then measured for the duration of the night phase (ZT12-24). In agreement with previous findings, control flies robustly suppressed sleep when starved on agar, while there was no significant effect of starvation on sleep duration in trsn^null flies (Figure 6A and B). In both control and trsn^null flies, CO₂ production was reduced during starvation, suggesting trsn is not required for modulating MR in accordance with feeding state. These findings fortify the notion that MR can be regulated independently from both sleep and locomotor activity (Figure 6C and D). Interestingly, while MR was further reduced in trsn^null flies upon starvation, the basal MR of fed trsn^null flies was lower than w¹¹¹⁸ controls (Figure 6C and D). For both w¹¹¹⁸ and trsn^null flies, there was a stronger relationship between MR and sleep bout duration in fed flies than starved flies (Figure 6E and F), fortifying the notion that sleep-dependent changes in MR are not disrupted in trsn^null flies. Together, these results indicate that trsn is required for starvation-induced sleep suppression but is dispensable for sleep-induced modulation of MR.

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.

DISCUSSION

MR is regulated in accordance with environmental changes and life history, providing a metric for whole-body metabolic function. While mammalian studies typically determine MR via O₂ consumption or respiratory quotient (ratio of CO_{2 eliminated}/O_{2 consumed}), studies in Drosophila commonly measure CO₂ production because it is directly correlated with O₂ input and accurately reflects MR.^14,16,30 Previous systems investigating MR in Drosophila have used single flies or populations to measure changes in CO₂ in response to aging, temperature change, and dietary restriction.^16,31,32 Here, we have modified a previously described single-fly respirometry system and DAM system to simultaneously measure MR and sleep. This system can measure CO₂ production and locomotor activity over a 24-hour period, providing the ability to measure the relationship between sleep and metabolism, providing a system to investigate the complex relationships between diverse genetic and environmental factors with MR.

Sleep-Metabolism Interactions in Mammals and Arthropods

Regulation of sleep and metabolism is conserved at the molecular and physiological levels between Drosophila and mammals.^33,34 Similar to mammals, flies modulate sleep and feeding in accordance with metabolic state, providing a system to investigate the genetic underpinnings of these behaviors.² For instance, when starved, both flies and mammals suppress sleep presumably to forage for food.^18,35,36 The finding that sleep-dependent reductions in MR are conserved in Drosophila supports the notion that an essential function of sleep is metabolic regulation. A number of previous studies suggest total sleep duration is positively correlated with basal MR, supporting the notion that sleep may be an adaptive mechanism of energy conservation.^37,38 However, a meta-analysis study examining over 40 different mammalian species revealed a negative relationship between sleep and basal MR, opposing the energy conservation model of sleep.³⁹ In humans, reduced MR during sleep accounts for as much as a 15% energy savings.^40–42 Our findings reveal a similar reduction of MR during sleep in fruit flies, suggesting this may provide an evolutionarily adaptive mechanism to conserve energy.

While our study is the first to examine the relationship between sleep and MR in Drosophila, previous studies implicated shared genetic or environmental factors in the regulation of sleep and metabolic function. For example, dopamine potently suppresses sleep in Drosophila, and flies harboring a mutation in the dopamine transporter gene fumin (fmn) exhibit reduced sleep and increased CO₂ production,^43,44 suggesting dopamine regulates both sleep and metabolic state. Importantly, MR remained elevated in fumin mutant flies when motor neurons were genetically silenced, indicating that the elevated MR does not result from differences in locomotor activity.⁴⁴ Moreover, long-term sleep deprivation in Pacific beetle cockroach, Diploptera punctate, caused significant increases in O₂ consumption and elevated basal MR compared to controls, indicating that sleep loss impacts metabolism.⁴⁵ These data are in agreement with our findings, where we identify increased MR during sleep deprivation and reduced MR during sleep rebound or pharmacologically induced sleep, revealing a fundamental and direct relationship between sleep and lower MR, indicating that changes in CO₂ production during sleep are due to changes in basal MR, rather than reduced locomotor activity.

Environmental Factors Regulating Sleep and Metabolism

In Drosophila, sleep and MR are influenced by diet, temperature, and age.^46,47 Here, we discover that starvation conditions impede the physiological changes associated with normal sleep. This simultaneous assessment of sleep and metabolic state can be applied to determine how MR and sleep are related to starvation resistance. Selection for starvation-resistant Drosophila through experimental evolution results in flies that can survive over 2 weeks without food and exhibit a host of metabolic and developmental differences, thus providing a system to examine interactions between metabolism and behavior.^48,49 The starvation-resistant flies exhibit increased body size, energy stores, and reduced MR, providing numerous mechanisms for energy conservation.^49–51 Previously, we reported that sleep duration is increased in starvation-resistant flies and proposed that this provides an additional mechanism for energy conservation.^50,52 Application of this approach measuring sleep and MR will provide the ability to determine MR in asleep and awake flies and identify whether reduced MR in starvation-resistant flies is a consequence of increased sleep or these traits have evolved in parallel.

Evidence for Sleep-Associated Regulation of MRs in Drosophila

In birds and mammals, sleep is associated with changes in cortical activity resulting in defined stages of sleep, such as rapid eye movement and nonrapid eye movement, which differ in physiology and function.^53,54 In Drosophila, sleep studies have primarily used behavioral quiescence and body postures to denote sleep, and much less is known about how sleep impacts physiology. Recording of local field potentials in tethered animals reveals distinct differences between quiescent and active states, and sleep is associated with a reduction in 15–30 Hz local-field potentials. The reduction in 15–30 Hz oscillations is at its greatest following 15 minutes of immobility, suggesting that this physiological change in neuronal activity represents a deeper form of sleep, along with coordinate increases in arousal threshold.^9,10 Evaluating sleep intensity in male and female Drosophila using an arousal-testing paradigm during extended nighttime sleep bouts identified a gradual decrease in responsiveness until a second, deeper sleep state was reached after ~30 minutes.^10,55 Consistent with these findings, we report that MR decreases with sleep duration, reaching a significant reduction ~30 minutes following sleep onset. Taken together, these findings suggest MR may provide a physiological indicator of sleep intensity that compliments existing electrophysiology and behavioral methods of analysis to define a deeper sleep state in flies.

A Role for translin in Regulating MR

The RNA-binding protein trsn is a proposed integrator of sleep and metabolic state, and flies deficient for trsn fail to suppress sleep in response to starvation.²⁹ Notably, the defect in trsn-mutant flies is specific to regulation sleep regulation because trsn-deficient flies have normal feeding and energy stores.²⁹ Here, we find that starvation reduces MR in trsn mutant and wild-type flies. Even though trsn mutants demonstrate starvation-induced sleep suppression, our findings indicate that MR can still be modulated in trsn mutants in a starved state. We identify a lower basal MR in fed trsn mutants compared to controls, though this may be attributable to the trend toward increased nighttime sleep in trsn mutant flies. In addition to trsn, a number of additional genes and transmitters have been identified as regulating starvation-induced sleep suppression or hyperactivity, including Octopamine, clock, and the glucagon-like adipokinetic hormone.^35,56,57 Therefore, this assay provides a direct readout of metabolic response to starvation and can be used for more detailed investigation of the mechanisms underlying the integration of sleep and metabolic state.

Future Applications for Investigation of MR in Drosophila

Beyond our initial analysis of the relationship between sleep and MR in Drosophila, this system allows for genetic screens or targeted genetic manipulations to identify novel genes regulating sleep, MR, and the integration of these processes. For example, the mushroom bodies, fan-shaped body, and circadian neurons modulate sleep and wakefulness in Drosophila,^23,58–61 and the effects of manipulating these systems on sleep-dependent modulation of MR could be measured using this system. Similarly, application of this system could identify novel genes, neurons or environmental factors required for changes in MR during sleep. Recent studies in flies have identified neural circuits involved in sleep homeostasis,^62–64 yet little is known about the physiological changes associated with rebound sleep in flies. In addition to sleep, the circadian system regulates metabolism in flies and mammals.^65,66

In addition to measuring MR, respirometry can also be used to measure specific molecules being metabolized. The simultaneous measurement of CO₂ and O₂ enables the ability to identify the exchange ratios of O₂ consumed and CO₂ produced and ultimately infer the specific energy fuels utilized.^67,68 More specifically, the ratio between the CO₂ produced and O₂ consumed at a steady state, also known as the respirometry quotient (RQ) or the respirometry exchange ratio which quantifies the same ratio but at any time point (eg, during exercise), can be used to identify food sources metabolized including fat (RQ = ~0.7), carbohydrates (RQ = ~1.0), or protein (RQ = ~0.8–0.9).¹⁴ Respirometry measurements have been used to identify substrates metabolized in both mammals^69,70 and invertebrates.¹⁶ However, the sensitivity of O₂ detection is lower than CO_2, thus preventing detection of O₂ changes in single flies, yet recent studies indicate that sleep can be measured in group-housed Drosophila.⁷¹ Therefore, it may be feasible for future studies utilizing groups of flies to determine metabolized energy stores using this system.

CONCLUSIONS

We describe a system for simultaneously measuring sleep and MR and, further, identify dynamic regulation of MR during individual sleep bouts. This system denotes MR as a readily identifiable marker of the physiological changes associated with sleep, which can be universally applied to examine the function of novel sleep genes and neurons in Drosophila. Ultimately, this unique system can be applied to examine precise interactions between numerous aspects of life history and circadian function coordinately with MR.

SUPPLEMENTARY MATERIAL

Supplementary material is available at SLEEP online.

FUNDING

This work was supported by NIH grants 1R01NS085252 to ACK and R15NS080155 to ACK and JRD.

DISCLOSURE STATEMENT

None declared.