Table 1.
Primer sequences for quantitative PCR.
Figure 1.
Mean food intake and S.E.M. of control, ARP, AAP, FRP and FAP rats during the day (white bars) and during the night (gray bars) during the fourth week of manipulation.
Asterisks indicate statistical difference between day and night values (P<0.05).
Figure 2.
Changes in metabolic parameters after 4 weeks activity in the rest phase.
Mean (±SEM) body weight gain (A,C), and abdominal fat weight (B,D) of control (n = 8) and rats exposed to forced activity during their rest phase (ARP) or during their activity phase (AAP) (n = 6 per group) and rats with restricted food during the rest phase (FRP) or during their activity phase (FAP) (n = 9 per group). All groups showed a similar daily pattern of food consumption); however, ARP and FRP rats increased their body weight (A,C) and attained a higher accumulation of abdominal fat (B,C) while AAP rats remained at similar values as controls. (P<0.001). Statistical analyses were performed with a Tukey multiple comparisons post hoc test.
Figure 3.
Glucose intolerance after 4 weeks of activity and or food in the rest phase.
Animals received at time 0 (basal) an intraperitoneal injection of 1 g of glucose per kg body weight. Glucose levels in ARP animals show immediately after the glucose injection an increase in plasma glucose levels that remained significant higher as compared to the AAP and CTRL group until 90 minutes after the challenge. Glucose levels in FRP animals show after the glucose injection a similar increase in plasma glucose levels; however at 60 minutes no difference between FAP and CTRL group can be noted.
Figure 4.
When rats are active and eat during the rest phase microvesicular steatosis were observed.
Oil-Red-O staining of frozen sections of livers obtained from CTRL, ARP and AAP rats. Mayer’s hematoxylin was used for counterstaining. In ARP rats were observed typical lipid microdroplets that are indicative of microvesicular steatosis. In L is shown the mean of the area of the red oil positive tissues,; (P<0.0001).
Figure 5.
Except for Per2 in ARP and FRP, clock genes in the liver follow food and activity.
Relative mRNA temporal profiles of clock genes along the light/dark (LD) cycle were examined in the liver of control. (n = 3 rats per time point). A) Black and white bars at the bottom of the figure represent the LD cycle; the striped bars represent the time spent in the slow rotating wheel for the ARP an AAP rats respectively. B) The striped bar indicates the light period in which FRP animals received their food. Quantification of genes was normalized to Actin. Daily curves for clock and metabolic genes were tested with a cosinor analysis to determine significance in amplitude and to determine the acrophase. See also table 2 for details. Asterisk indicates a significant rhythm in the CTRL group, † indicates a significant rhythm in the ARP group, # indicates a significant rhythm in the FRP group,+indicates a significant rhythm in the AAP group and ‡ indicates significant rhythm in the FAP group. These results were corroborated with Kruskall-Wallis test (Table 3).
Table 2.
Cosinor analysis of clock and metabolic gene expression.
Table 3.
Kruskall-Wallis test. P>0.05 was considered not rhythmic.
Figure 6.
Nampt and NAD+ lose their rhythmicity in rats eating during the rest phase.
Relative expression levels of Nampt mRNA,and NAD+ mRNA in liver of CTRL, ARP, FRP, AAP and FAP rats during the 24-h LD cycle. Each value represents the mean ± SEM (n = 3–4 per time point). Other indications as in Fig. 5.
Figure 7.
When rats are active and eat during the rest phase metabolic genes are down regulated.
Relative expression levels of Sirt1 mRNA and SIRT1 protein in liver of CTRL, ARP, FRP, AAP and FAP rats are presented during the 24-h LD cycle. Each value represents the mean ± SEM (n = 3–4 per time point). Other indications as in Fig. 5.
Figure 8.
PGC-1α and Ppar genes show a loss of rhythm and are down regulated when rats eat during the rest phase.
Relative expression levels of Pgc-1α, Pparα and Pparγ mRNA in liver of CTRL, ARP, FRP, AAP and FAP rats, during the 24-h LD cycle (n = 3–4 per time point). Each value represents the mean ± SEM. Other indications as in Fig. 5.