Ected for measurement of triglycerides, FFAs, and ketone body levels. Plasma triglycerides and FFAs were determined by GPO-HDAOS (triglycerides) and ACS-ACOD (FFAs) enzyme assays using 10781694 an automatic biochemical analyzer system (HITACHI 7180, Hitachi, Tokyo, Japan). Ketone bodies (b-hydroxybutyrate and acetoacetate) were measured by an automatic analyzer system JCA-BM12 (JEOL, Tokyo, Japan) using reagents for measurement of ketoneIn vivo Metabolic TestingGlucose tolerance was assessed after glucose intraperitoneal (i.p.) injection (2 g/kg for mice aged 14 weeks) in unrestrained awake mice after a 16-hour fast. Insulin tolerance tests (1 unit/kg for mice aged 14 weeks, Sigma Chemical Co., St. Louis, MO, USA) were performed in mice after a 6-hour fast (ZT6).Augmented Sleep Pressure Model in MiceFigure 3. The influence of dietary Autophagy restriction during gestation on sleep homeostasis in adult offspring mice. Power spectral analysis of EEG during NREM sleep (A). Hourly time course changes of EEG delta/theta ratio in NREM sleep (B), and the averages for each 6-hour period (C) across ZT0-6 (L1), ZT6-12 (L2), ZT12-18 (D1), and ZT18-24 (D2). Six-hour changes of the rebound rate of delta/theta ratios after sleep deprivation (D). Open bars and circles indicate AD mice. Closed bars and circles indicate DR mice. Data represent means 6 SEM (A 16985061 ; n = 6). **p,0.01 and *p,0.05 indicate a significant difference. doi:10.1371/journal.pone.0064263.gReal Time RT-PCR inhibitor AnalysisFor molecular analyses, fetal mice were sacrificed at ZT9-10, and then liver and whole brain were extracted at gestation day 17. Adult offspring mice at the age of 8? weeks were sacrificed at ZT4-5, and then liver and brain were extracted. The brain was sectioned coronally on ice with a brain slicer (Muromachi Kikai, Tokyo, Japan). Coronal brain sections were divided into fractions of hypothalamus, cerebral cortex, hippocampus, and striatum by a brain matrix. Brain and liver tissue were immediately frozen in liquid nitrogen, and stored at 280uC until use. Total RNA in fetal and adult offspring mice was isolated following Takara’s RNA isolation protocol (RNAiso Plus; Takara Bio, Shiga, Japan). cDNA in fetal and adult offspring mice was generated from each RNA sample using a High-Capacity cDNA Transcription Kit (Applied Biosystems, Foster, CA, USA). We used predesigned, gene-specific TaqMan probes and primer sets to assess expression of the genes indicated in Table S1. Real-time PCR was performed with an Applied Biosystems 7900HT real-time PCR system using TaqMan Universal PCR Master Mix (Roche Applied Science, Mannheim, Germany) according to the manufacturer’s instructions. Cytoplasmic beta-actin (b-actin, encoded by Actb) was used for anendogenous quantitative control, and values were normalized to b-actin mRNA expression.Pharmacological Treatments and Injection ProceduresTo investigate the effect of caffeine on behavior, caffeine (15 mg/kg, Sigma Chemical Co) was administered i.p. 30 min before the forced swim test. The detailed procedure of the forced swim test is described in Protocol S1. In order to evaluate the effect of caffeine on sleep, caffeine (5 mg/kg) was injected at ZT0 during sleep recordings. The caffeine dose was selected according to a previous study [27].StatisticsResults are expressed as means 6 SEM. Changes in body weight, body temperature, spontaneous activity, sleep architecture and EEG delta/theta ratio were analyzed by repeated measures one-way or two-way analysis of vari.Ected for measurement of triglycerides, FFAs, and ketone body levels. Plasma triglycerides and FFAs were determined by GPO-HDAOS (triglycerides) and ACS-ACOD (FFAs) enzyme assays using 10781694 an automatic biochemical analyzer system (HITACHI 7180, Hitachi, Tokyo, Japan). Ketone bodies (b-hydroxybutyrate and acetoacetate) were measured by an automatic analyzer system JCA-BM12 (JEOL, Tokyo, Japan) using reagents for measurement of ketoneIn vivo Metabolic TestingGlucose tolerance was assessed after glucose intraperitoneal (i.p.) injection (2 g/kg for mice aged 14 weeks) in unrestrained awake mice after a 16-hour fast. Insulin tolerance tests (1 unit/kg for mice aged 14 weeks, Sigma Chemical Co., St. Louis, MO, USA) were performed in mice after a 6-hour fast (ZT6).Augmented Sleep Pressure Model in MiceFigure 3. The influence of dietary restriction during gestation on sleep homeostasis in adult offspring mice. Power spectral analysis of EEG during NREM sleep (A). Hourly time course changes of EEG delta/theta ratio in NREM sleep (B), and the averages for each 6-hour period (C) across ZT0-6 (L1), ZT6-12 (L2), ZT12-18 (D1), and ZT18-24 (D2). Six-hour changes of the rebound rate of delta/theta ratios after sleep deprivation (D). Open bars and circles indicate AD mice. Closed bars and circles indicate DR mice. Data represent means 6 SEM (A 16985061 ; n = 6). **p,0.01 and *p,0.05 indicate a significant difference. doi:10.1371/journal.pone.0064263.gReal Time RT-PCR AnalysisFor molecular analyses, fetal mice were sacrificed at ZT9-10, and then liver and whole brain were extracted at gestation day 17. Adult offspring mice at the age of 8? weeks were sacrificed at ZT4-5, and then liver and brain were extracted. The brain was sectioned coronally on ice with a brain slicer (Muromachi Kikai, Tokyo, Japan). Coronal brain sections were divided into fractions of hypothalamus, cerebral cortex, hippocampus, and striatum by a brain matrix. Brain and liver tissue were immediately frozen in liquid nitrogen, and stored at 280uC until use. Total RNA in fetal and adult offspring mice was isolated following Takara’s RNA isolation protocol (RNAiso Plus; Takara Bio, Shiga, Japan). cDNA in fetal and adult offspring mice was generated from each RNA sample using a High-Capacity cDNA Transcription Kit (Applied Biosystems, Foster, CA, USA). We used predesigned, gene-specific TaqMan probes and primer sets to assess expression of the genes indicated in Table S1. Real-time PCR was performed with an Applied Biosystems 7900HT real-time PCR system using TaqMan Universal PCR Master Mix (Roche Applied Science, Mannheim, Germany) according to the manufacturer’s instructions. Cytoplasmic beta-actin (b-actin, encoded by Actb) was used for anendogenous quantitative control, and values were normalized to b-actin mRNA expression.Pharmacological Treatments and Injection ProceduresTo investigate the effect of caffeine on behavior, caffeine (15 mg/kg, Sigma Chemical Co) was administered i.p. 30 min before the forced swim test. The detailed procedure of the forced swim test is described in Protocol S1. In order to evaluate the effect of caffeine on sleep, caffeine (5 mg/kg) was injected at ZT0 during sleep recordings. The caffeine dose was selected according to a previous study [27].StatisticsResults are expressed as means 6 SEM. Changes in body weight, body temperature, spontaneous activity, sleep architecture and EEG delta/theta ratio were analyzed by repeated measures one-way or two-way analysis of vari.
