Ance (ANOVA) followed by Scheffe’s post-hoc test. The results of latency for awaking following cage shaking and latency to sleep following a caffeine injection were analyzed by unpaired Student’s t-test. The results of behavioral tests, real-time RT-PCR, blood glucose, and measureAugmented Sleep Pressure Model in MiceFigure 4. Threshold for waking by external stimuli (cage shaking) in adult offspring mice. Photos of the experimental setting for estimating waking threshold in mice against external sensory stimuli (A). Cage shaking was started 30 seconds after the continuous appearance of EEG delta waves as shown in upper traces. We measured the latency from the start of shaking to EEG arousal. The latency for waking following shaking stimuli (B). Open bars indicate AD mice. Closed bars indicate DR mice. Data represent means 6 SEM (B; n = 6). **p,0.01 indicates a Title Loaded From File significant difference. doi:10.1371/journal.pone.0064263.gments of plasma substances were analyzed by Mann-Whitney U test. P,0.05 was assumed to indicate statistical significance.Results Body WeightThe dietary restriction (DR) female mice showed significantly less body weight gain during the 4 days before parturition and just after parturition (Figure S1A). DR female mice displayed a marked decrease in blood glucose (Figure S1B). However, there were no significant 16985061 differences in the number of either live births or dead births (Figure S1C, D). The ratio of males to Title Loaded From File females was not significantly different between control (fed ad libitum; AD) and DR offspring mice (Figure S1E). DR offspring mice exhibited significantly reduced body weights at birth (Figure 1A). However, as early as the third postnatal day, the significant differences inbody weight had already disappeared (Figure 1B). That is, the DR mice exhibited LBW accompanied by an accelerated catch-up growth (CUG). Up to 8 weeks of age, when the sleep recordings and behavioral tests were carried out, no significant differences were observed in body weight between the two groups (Figure 1C).Sleep Architecture and HomeostasisNo significant changes were observed in the diurnal pattern and amount of wake, NREM, and REM sleep between the two groups (Figure 2A ). Additionally, mean bin size, number of episodes, and mean interval of sleep/wake cycles in DR mice were also not changed (Figure 2D ). The body temperature and its diurnal variation in DR mice were not largely modified (Figure 2G), partially and indirectly suggesting normal thermoregulation and/ or circadian rhythmicity. In comparison, DR mice displayed lower spontaneous activity, especially in the first half of the dark periodFigure 5. Metabolic state in fetal stage (gestation day 17). Blood glucose (A) and gene expression related to the regulation of lipid metabolism in liver (B) and whole brain (C). Open bars indicate AD mice. Closed bars indicate DR mice. Data represent means 6 SEM (A ; n = 6). **p,0.01 indicates a significant difference. doi:10.1371/journal.pone.0064263.gAugmented Sleep Pressure Model in MiceFigure 6. Metabolic state in adulthood (8? weeks). The plasma levels of triglycerides (A), free fatty acids (FFAs; B), b-hydroxybutyrate (C), acetoacetate (D), and ketone bodies (E). Glucose tolerance test (GTT; F) and insulin tolerance test (ITT; G) in adulthood. Gene expression related to the regulation of lipid metabolism in liver (H) and hypothalamus (I). Open bars and circles indicate AD mice. Closed bars and circles indicate DR mice. Data represent means 6 SEM.Ance (ANOVA) followed by Scheffe’s post-hoc test. The results of latency for awaking following cage shaking and latency to sleep following a caffeine injection were analyzed by unpaired Student’s t-test. The results of behavioral tests, real-time RT-PCR, blood glucose, and measureAugmented Sleep Pressure Model in MiceFigure 4. Threshold for waking by external stimuli (cage shaking) in adult offspring mice. Photos of the experimental setting for estimating waking threshold in mice against external sensory stimuli (A). Cage shaking was started 30 seconds after the continuous appearance of EEG delta waves as shown in upper traces. We measured the latency from the start of shaking to EEG arousal. The latency for waking following shaking stimuli (B). Open bars indicate AD mice. Closed bars indicate DR mice. Data represent means 6 SEM (B; n = 6). **p,0.01 indicates a significant difference. doi:10.1371/journal.pone.0064263.gments of plasma substances were analyzed by Mann-Whitney U test. P,0.05 was assumed to indicate statistical significance.Results Body WeightThe dietary restriction (DR) female mice showed significantly less body weight gain during the 4 days before parturition and just after parturition (Figure S1A). DR female mice displayed a marked decrease in blood glucose (Figure S1B). However, there were no significant 16985061 differences in the number of either live births or dead births (Figure S1C, D). The ratio of males to females was not significantly different between control (fed ad libitum; AD) and DR offspring mice (Figure S1E). DR offspring mice exhibited significantly reduced body weights at birth (Figure 1A). However, as early as the third postnatal day, the significant differences inbody weight had already disappeared (Figure 1B). That is, the DR mice exhibited LBW accompanied by an accelerated catch-up growth (CUG). Up to 8 weeks of age, when the sleep recordings and behavioral tests were carried out, no significant differences were observed in body weight between the two groups (Figure 1C).Sleep Architecture and HomeostasisNo significant changes were observed in the diurnal pattern and amount of wake, NREM, and REM sleep between the two groups (Figure 2A ). Additionally, mean bin size, number of episodes, and mean interval of sleep/wake cycles in DR mice were also not changed (Figure 2D ). The body temperature and its diurnal variation in DR mice were not largely modified (Figure 2G), partially and indirectly suggesting normal thermoregulation and/ or circadian rhythmicity. In comparison, DR mice displayed lower spontaneous activity, especially in the first half of the dark periodFigure 5. Metabolic state in fetal stage (gestation day 17). Blood glucose (A) and gene expression related to the regulation of lipid metabolism in liver (B) and whole brain (C). Open bars indicate AD mice. Closed bars indicate DR mice. Data represent means 6 SEM (A ; n = 6). **p,0.01 indicates a significant difference. doi:10.1371/journal.pone.0064263.gAugmented Sleep Pressure Model in MiceFigure 6. Metabolic state in adulthood (8? weeks). The plasma levels of triglycerides (A), free fatty acids (FFAs; B), b-hydroxybutyrate (C), acetoacetate (D), and ketone bodies (E). Glucose tolerance test (GTT; F) and insulin tolerance test (ITT; G) in adulthood. Gene expression related to the regulation of lipid metabolism in liver (H) and hypothalamus (I). Open bars and circles indicate AD mice. Closed bars and circles indicate DR mice. Data represent means 6 SEM.
