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Changes in components of energy regulation in mouse cortex with increases in wakefulness - PubMed

Changes in components of energy regulation in mouse cortex with increases in wakefulness

Elena V Nikonova et al. Sleep. 2010 Jul.

Abstract

Study objectives: Increases in ATP production machinery have been described in brain after 3 h of sleep deprivation. Whether this is sustained with longer durations of extended wakefulness is unknown. We hypothesized that energy depletion could be a mechanism leading to difficulty maintaining wakefulness and assessed changes in components of the electron transport chain.

Design: Protein levels of key subunits of complexes IV and V of the electron transport chain (COXI, COXIV, ATP5B) and uncoupling protein 2 (UCP2) in isolated mitochondria by Westerns in mouse cerebral cortex after 3 and 12 h of sleep deprivation were compared to that in control mice. Activity of complex IV enzyme and relevant transcription factors-Nrf1, Nrf2 (Gabp), and phosphorylation of AMP-dependent kinase (AMPK)-were also assessed.

Participants: 8-10 week old C57BL/6J male mice (n = 91).

Interventions: 3, 6, and 12 h of sleep deprivation.

Measurements and results: After both 3 and 12 h of sleep deprivation, complex IV proteins and enzyme activity were significantly increased. The complex V catalytic subunit was significantly increased after 12 h of sleep deprivation only. Increased levels of UCP2 protein after 12 h of sleep deprivation suggests that there might be alterations in the ATP/AMP ratio as wakefulness is extended. That phosphorylation of AMPK is increased after 6 h of sleep deprivation supports this assertion. The increase in Nrf1 and Nrf2 (Gabp) mRNA after 6 h of sleep deprivation provides a mechanism by which there is up-regulation of key proteins.

Conclusions: There are complex dynamic changes in brain energy regulation with extended wakefulness.

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Figures

Figure 1
Figure 1

Representative immunoblots of OXPHOS proteins–COXI, COXIV, and ATP5B–after 3 h (panel A) and 12 h (panel B) of spontaneous (undisturbed) sleep (3C, 12C) and sleep deprivation (3SD, 12SD). Each blot shows a single biological replicate run in duplicate. 3-6 gels each had a sleep deprived group and a spontaneous sleep group and were run using 2-3 biological replicates per group in duplicate. Normalization was done using the expression of target protein (COXI, COXIV, ATP5B) in a whole brain mitochondria sample run on every gel.

Figure 2
Figure 2

Effects of 3 and 12 h of sleep deprivation on protein level of OXPHOS subunits in mouse cerebral cortex: (A) mitochondrial catalytic COXI subunit of complex IV; (B) nuclear COXIV subunit of complex IV; and (C) nuclear catalytic ATP5B subunit of complex V. Data are mean and standard deviation of fold change between groups (n = 6-8 per group). Left to right: after 3 h of sleep deprivation (3SD) compared to 3 h of spontaneous sleep (3C, grey bar); after 12 h of sleep deprivation (12SD) compared to undisturbed matching control (12C, dark grey bar); and after 3 h of sleep deprivation (3SD) compared to 3 h of spontaneous wake (3SW, black bar). COXI, COXIV protein levels are significantly increased after both 3 and 12 h of sleep deprivation compared to undisturbed diurnal controls while ATP5B level is significantly increased only after 12 h of sleep deprivation. Note that COXIV protein expression is significantly lower after 3 h of sleep deprivation compared to 3 h of spontaneous wake. Legend: 3SD–3 h of sleep deprivation (lights on); 3C–3 h of spontaneous sleep (lights on); 12SD–12 h of sleep deprivation (lights on); 12C–12 h after lights on (12 h spontaneous sleeping group); 3SW–3 h of spontaneous wake (lights off); *P < 0.05; **P < 0.01; ***P < 0.001

Figure 3
Figure 3

Enzymatic activity of OXPHOS complex IV (COX) increases with wakefulness in mouse cortex. Data are mean and standard deviations of absolute values measured per 1 μg protein from isolated mitochondria. Left to right: COX activity for mice sacrificed at 07:00 (lights on, n = 7); after 3 h of spontaneous sleep (3C, n = 6); after 3 h of spontaneous wake (3SW, n = 8); after 3 h of sleep deprivation (3SD, n = 6); and 12 h of sleep deprivation (12SD, n = 6). There is a significant increase in COX activity after 3 h of sleep deprivation (3SD) compared to 3 h of spontaneous sleep (3C). No significant changes in COX activity were found between 3 (3SD) and 12 (12SD) h of sleep deprivation. The time of sacrifice is given below each bar. H indicates groups that were sleep deprived by handling. Legend: 07:00–time zero (lights on); 3C–3 h of spontaneous sleep (lights on); 3SW–3 h of spontaneous wake (lights off); 3SD–3 h of sleep deprivation (lights on); 12SD–12 h of sleep deprivation (lights on); **P = 0.011.

Figure 4A
Figure 4A

UCP2 protein level increases after 12 h of sleep deprivation in mouse cerebral cortex. Representative immunoblot for UCP2 containing samples collected after 12 h of undisturbed lights on period (12C, lanes 1-3); 12 h of sleep deprivation (12SD, lanes 4-6), and whole brain mitochondria (WB, lanes 7-8).

Figure 4B
Figure 4B

UCP2 protein level increases after 12 h of sleep deprivation in mouse cerebral cortex. Data on the left represent fold change in UCP2 protein expression level calculated using the means between the corresponding groups as for OXPHOS proteins. Left to right: fold change after 3 h of sleep deprivation (3SD) compared to 3 h of spontaneous sleep (3C, light grey bar); after 12 h of sleep deprivation (12SD) compared to undisturbed diurnal control (12C, medium grey bar), and after 3 h of sleep deprivation (3SD) compared to 3 h of spontaneous wake (3SW, black bar). Data on the right represent the whole brain mitochondria normalized UCP2 protein expression (mean ± standard deviation, n = 8-16). Animals were sacrificed 3 or 12 h after lights on, those that were sleep deprived (handled) are shown in gradient grey, and their undisturbed matching controls by open bars. Left to right: 3 h of sleep deprivation (3SD); 12 h of sleep deprivation (12SD); 3 h of spontaneous sleep (3C); and 12 h of lights on period (12C). There is a significant increase in UCP2 protein level after 12 h of sleep deprivation (12SD) compared to both undisturbed diurnal control (12C, P = 0.024) and 3 h of sleep deprivation (3SD, P = 0.04). *P < 0.05.

Figure 5
Figure 5

Temporal changes in the expression of the Nrf1 (A), and Nrf2 (Gabpa) (B) genes in the mouse cerebral cortex. The expression profiles were established after 3, 6, 9, and 12 h of spontaneous sleep denoted in the figure as control (squares/dashed lines) or sleep deprivation (triangles/solid lines). The mRNA level of the Nrf1 and Nrf2 genes increases during sleep deprivation and does not change or decline during sleep. A false discovery rate (FDR) was calculated for each gene as described by Storey to determine how significant the differences are in gene expression between sleep and sleep deprivation. The FDR values for the Nrf1 and Nrf2 genes is estimated at 1.3 × 10−4 and 1 × 10−5, respectively. Transcript abundance change is expressed in arbitrary units, and is log2 of the mean fluorescence signal for each time point minus that of the control animals sacrificed at 07:00 or time “0”. The data presented in this figure originate from a recently completed microarray study.

Figure 6
Figure 6

Changes in the steady-state transcript levels of Nrf1, Nrf2 (Gabpa) and Pgc1a in the cerebral cortex of C57BL/6J mice after 6 h of total sleep deprivation and 6 h of spontaneous sleep. The transcript level at time zero (light on at 07:00) was used for calibration of other groups. Sleep deprivation significantly up-regulates the expression of both Nrf1 and Nrf2 (Gabpa) genes (P < 0.0001). These results are in agreement with previously published microarray data. The Pgc1a transcript level in cerebral cortex did not change with sleep deprivation (P = 0.764). Legend: 6SD–6 h of sleep deprivation; 6C–6 h of undisturbed sleep; ***P < 0.0001.

Figure 7
Figure 7

Assessment of α-AMPK and phosphorylated α-AMPK following immunoprecipitation for (A) α-1 AMPK and (B) α-2 AMPK. Data are presented as average ± SEM (n = 9). α-AMPK and phosphorylated α-AMPK (P-α1-AMPK) are placed on different y-axes. There is a significant increase in phosphorylated α-2 AMPK in sleep deprived samples, *P < 0.05.

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