Time-restricted feeding reveals a role for neural respiratory clocks in optimizing daily ventilatory-metabolic coupling in mice

Abstract

The master circadian clock, located in the suprachiasmatic nuclei (SCN), organizes the daily rhythm in minute ventilation (V̇e). However, the extent that the daily rhythm in V̇e is secondary to SCN-imposed O₂ and CO₂ cycles (i.e., metabolic rate) or driven by other clock mechanisms remains unknown. Here, we experimentally shifted metabolic rate using time-restricted feeding (without affecting light-induced synchronization of the SCN) to determine the influence of metabolic rate in orchestrating the daily V̇e rhythm. Mice eating predominantly at night exhibited robust daily rhythms in O₂ consumption (V̇o₂), CO₂ production (V̇co₂), and V̇e with similar peak times (approximately ZT18) that were consistent with SCN organization. However, feeding mice exclusively during the day separated the relative timing of metabolic and ventilatory rhythms, resulting in an approximately 8.5-h advance in V̇co₂ and a disruption of the V̇e rhythm, suggesting opposing circadian and metabolic influences on V̇e. To determine if the molecular clock of cells involved in the neural control of breathing contributes to the daily V̇e rhythm, we examined V̇e in mice lacking BMAL1 in Phox2b-expressing respiratory cells (i.e., BKOP mice). The ventilatory and metabolic rhythms of predominantly night-fed BKOP mice did not differ from wild-type mice. However, in contrast to wild-type mice, exclusive day feeding of BKOP mice led to an unfettered daily V̇e rhythm with a peak time aligning closely with the daily V̇co₂ rhythm. Taken together, these results indicate that both daily V̇co₂ changes and intrinsic circadian time-keeping within Phox2b respiratory cells are predominant orchestrators of the daily rhythm in ventilation.NEW & NOTEWORTHY The master circadian clock organizes the daily rhythm in ventilation; however, the extent that this rhythm is driven by SCN regulation of metabolic rate versus other clock mechanisms remains unknown. We report that metabolic rate alone is insufficient to explain the daily oscillation in ventilation and that neural respiratory clocks within Phox2b-expressing cells additionally optimize breathing. Collectively, these findings advance our mechanistic understanding of the circadian rhythm in ventilatory control.

Keywords: BMAL1; Phox2b; breathing; circadian rhythm; metabolic rate.

Graphical abstract

Figure 1.

Time-restricted feeding protocol. Wild-type C57Bl6/J mice were randomly distributed into four TRF groups: chow-fed ad libitum (Chow), HFD-fed ad libitum (HFD), HFD night-fed, or HFD day-fed, and maintained on their respective feeding schedules for 4 wk. Ventilation was assessed during weeks 2 and 3 of TRF using whole body plethysmography. Metabolic rate was assessed during weeks 3 and 4 of TRF using indirect calorimetry. TRF, time-restricted feeding. Created using Biorender.com.

Figure 2.

Day feeding shifts rhythms in metabolic rate toward the timing of food intake. Under chow-fed and HFD-fed conditions, V̇o₂ (A) and V̇co₂ (B) exhibited a significant daily rhythm. Under day-fed and night-fed conditions, V̇o₂ (C) and V̇co₂ (D) exhibited a significant daily rhythm. Mice exhibited significant day-night differences in V̇o₂ (E) and V̇co₂ (F) under all feeding conditions except day-fed. n = 6 for all groups (three male, three female). A–D: Cosinor fit analysis where α = 0.05. E and F: repeated measures two-way ANOVA with Sidak’s post hoc. *P < 0.05, **P < 0.01, ****P < 0.0001. HFD, high-fat diet; DF, day-fed; NF, night-fed; ns, not significant; V̇co₂, carbon dioxide production; V̇o₂, oxygen consumption; ZT, zeitgeber time.

Figure 3.

The daily rhythm in minute ventilation is disrupted under exclusive day feeding. A: minute ventilation exhibited a significant daily rhythm under chow-fed and HFD-fed conditions. B: minute ventilation exhibited a significant daily rhythm under night-fed but not day-fed conditions. C: the daily mean minute ventilation of night-fed mice was increased relative to chow-fed mice. Day-fed mice failed to exhibit significant day-night variation in minute ventilation. n = 8 for all groups (four males, four females). A and B: Cosinor fit analysis where α = 0.05. C: one-way ANOVA with Tukey’s post hoc (left) and repeated measures two-way ANOVA with Sidak’s post hoc (right). *P < 0.05, **P < 0.01, ****P < 0.0001. HFD, high-fat diet; DF, day-fed; NF, night-fed; ZT, zeitgeber time.

Figure 4.

The daily rhythm in minute ventilation persists in BKOP mice fed exclusively during the day. A: BKOP mice exhibited a significant daily rhythm and day-night variation in V̇o₂ under both feeding conditions. BKOP mice exhibited a significant daily rhythm in V̇co₂ (B) and minute ventilation (C) under both feeding conditions and significant day-night variation under just the night-fed condition. n = 7 for all groups (five male, two female). A–C: Cosinor fit analysis where α = 0.05 (left) and repeated measures two-way ANOVA with Sidak’s post hoc (right). *P < 0.05, **P < 0.01, ****P < 0.0001. BKOP, BMAL1 knocked out of Phox2b cells, DF, day-fed; NF, night-fed; V̇co₂, carbon dioxide production; V̇o₂, oxygen consumption; ZT, zeitgeber time; ns, not significant.

Figure 5.

Day feeding differentially organizes the daily rhythm in minute ventilation of wild-type and BKOP mice. A: under all feeding conditions except day-fed, the phases of food intake, V̇o₂, and V̇co₂ (n = 6; three males, three females) in wild-type mice remained tightly aligned with the V̇e rhythm (n = 8; four males, four females) during the mid-dark phase. Under day feeding, V̇co₂ shifted toward food intake (P < 0.01). B: in night-fed BKOP mice, the phases of food intake, V̇o₂, V̇co₂, and V̇e rhythms remained tightly aligned to mid-dark phase. Under day feeding, the phase of V̇e was significantly different from that of V̇o₂ (P = 0.0083) but not V̇co₂ (P = 0.18). n = 7 for BKOP mice (five males, two females). A and B: two-way ANOVA with Tukey’s post hoc. BKOP, BMAL1 knocked out of Phox2b cells; DF, day-fed; NF, night-fed; V̇co₂, carbon dioxide production; V̇e, minute ventilation; V̇o₂, oxygen consumption; WT, wild-type; ZT, zeitgeber time.