Figure 1
Serum corticosterone and PER1 protein levels are entrained by food access. Diurnal corticosterone levels in rats fed ad libitum (AL) and under RF are shown in panel A. Quantitative analysis of clock protein PER1 using 50 μg of cytosolic fraction from at least 4 individuals is represented in panel B. Each value was normalized using the housekeeping protein actin as reference, and a representative western blot for each condition is shown. Black circles correspond to AL and white circles to RF group. The light gray rectangle above the x-axis represents mealtime for the food restricted group (ZT4-ZT6); * (p < 0.05) means significant difference among AL group time points and + (p < 0.05) significant difference among RF group time points (1-way ANOVA). # (p < 0.05) significant difference between AL vs RF (2-way ANOVA).
Table 1
Daily rhythm characteristics of plasma corticosterone and PER1 from rats under free access meal and restricted schedule determined by COSINOR test
| AL | RF | AL | RF | AL | RF | AL | RF | |
|---|---|---|---|---|---|---|---|---|
| PER1 (WB) | 17:30 ± 00:19 | 05:06 ± 00:24* | 0.27 ± 0.015 | 0.22 ± 0.011 | 0.55 ± 0.07 | 0.64 ± 0.06 | 0.002 | 0.04 |
The Acrophase, amplitude, and MESOR as well as the rhythmicity significance were determined as described in the Methods section. The values were determined from the diurnal profiles of these parameters. The units corresponding to amplitude and MESOR are mg/dl (corticosterone) and normalized units (PER1/ACTIN Western Blot). * (p < 0.05) significant between AL vs RF (Student´s t-test). Not significant 24 h rhythmicity was designed as NS.
Figure 2
Intracellular calcium-releasing channel binding assay. Binding activity of IP3R and RyR was measured as described in Methods. Panels A and C show daily patterns of the binding activity calculated for the IP3R in the ER and PM fractions, respectively. Panels B and D compare the feeding condition groups (Fasted and Refed) for IP3R binding in the ER and PM fractions, respectively. Panel E shows the daily pattern of the RyR binding activity. Panel F compares feeding condition groups for RyR-binding activity. Black circles correspond to AL, and white circles to RF group. The light gray rectangle above x-axis indicates meal time for the food restricted group (ZT4-ZT6). Mean values for four separate experiments are shown. Each data point was carried out in triplicate. * (p < 0.05) significant difference between AL time points and + (p < 0.05) significant difference in the RF group between their time points (1-way ANOVA); # (p < 0.05) significant between AL vs RF (2-way ANOVA). ♦ (p < 0.05) significant difference between Fasted and Refed groups; x (p < 0.05) significant difference between Fasted and RF-ZT3; and & (p < 0.05) significant difference between Refed and RF-ZT6 (Student´s t-test).
Table 2
Characteristics of the diurnal rhythms of hepatic calcium-handling proteins from rats under free access meal and restricted schedule determined by COSINOR test
| AL | RF | AL | RF | AL | RF | AL | RF | ||
|---|---|---|---|---|---|---|---|---|---|
| PMCA4 (WB) | 12:48 + 00:32 | 17:37 + 00:24* | 0.24 + 0.04 | 0.3 + 0.05 | 0.66 + 0.08 | 0.53 + 1.0 | 0.06NS | 0.04 |
The Acrophase, amplitude, and MESOR as well as the rhythmicity significance were determined as described in the methods section. Values were determined from the diurnal profiles of the parameters mentioned. The units corresponding to amplitude and MESOR are: fmoles/mg for IP3R and RyR binding assays; NADH/min/mg of protein for SERCA and PMCA activity; and normalized units for IP3R1-2, RyR and PMCA1-4 Western Blot (WB). * (p < 0.05) significant between AL vs RF (Student´s t-test). Not significant 24 h rhythmicity was designed as NS.
Figure 3
Detection of hepatic IP3Rs and RyR. The protein content of the calcium-release channels was evaluated by western blot (IP3Rs) and SDS-PAGE (RyR). Representative signals of IP3R1, IP3R2, and RyR and loading control (actin), and the daily rhythm profiles are shown in panels A, C, and E, respectively. Panels B, D, and F compare control groups of feeding condition (Fasted and Refed) for expression of the IP3R1, IP3R2, and RyR, respectively. Meanwhile IP3R1 and 2 were determined by conventional western blot, RyR protein was detected and quantified by stains-all methodology as described previously. Mean values for at least 4 independent experiments are shown. For IP3R type 1 and RyR proteins, 100 μg of the ER fraction was used. The plasma membrane fraction was used for IP3R type 2 (100 μg). Black circles correspond to AL and white circles to RF group. The light gray rectangle above x-axis indicates mealtime for the food restricted group (ZT4-ZT6). Mean values for at least 4 independent experiments are shown. Each data point was measured in triplicate. * (p < 0.05) significant difference between AL time points and + (p < 0.05) significant difference in the RF group between their time points (1-way ANOVA); # (p < 0.05) significant between AL vs RF (2-way ANOVA). ♦ (p < 0.05) significant difference between Fasted and Refed groups; x (p < 0.05) significant difference between Fasted and RF-ZT3; and & (p < 0.05) significant difference between Refed and RF-ZT6 (Student´s t-test).
Figure 4
Hepatic SERCA and PMCA activities Activity of Ca2+-ATPase in microsomal and plasma membrane fractions was measured as described in the methods. The daily profile of SERCA and PMCA activities are shown in panel A and C, respectively. Black circles correspond to AL, and white circles to RF group. The light gray rectangle above x-axis indicates mealtime for the food restricted group (ZT4-ZT6). The corresponding comparisons between feeding conditions (Fasted and Refed) are showed in panel B for SERCA activity and in panel D for PMCA activity. Mean values of 4 independent experiments are shown. Each data point was measured in triplicate. + (p < 0.05) significant difference in the RF group between their time points (1-way ANOVA); # (p < 0.05) significant between AL vs RF (2-way ANOVA). x (p < 0.05) significant difference between Fasted and RF-ZT3 and & (p < 0.05) significant difference between Refed and RF-ZT6 (Student´s t-test).
Figure 5
Western blots of hepatic SERCA and PMCA. The daily rhythm profiles and representative western blots of the Ca2+-ATPases SERCA2, PMCA1, and PMCA4 are shown in panels A, C, and E, respectively. Comparisons among the different feeding conditions are shown in panels B, D, and F for expression of SERCA2, PMCA1, and PMCA4, respectively. Mean values for at less 4 independent experiments are shown. For SERCA2 protein, 100 μg of ER fraction was used. The plasma membrane fraction (100 μg) was used in the case of PMCA type 1 and 4 proteins. Black circles correspond to AL, and white circles to RF group. The light gray rectangle above x-axis indicates mealtime for the food restricted group (ZT4-ZT6). Mean values for at least 4 independent experiments are shown. Each data point was measured in triplicate. + (p < 0.05) significant difference in the RF group between their time points (1-way ANOVA); # (p < 0.05) significant between AL vs RF (2-way ANOVA). x (p < 0.05) significant difference between Fasted and RF-ZT3 (Student´s t-test).
Figure 6
Zonal distribution of hepatic calcium-handling proteins. Zonal distribution of IP3R type 1 (panel A), IP3R type 2 (panel B), ryanodine receptor (panel C), SERCA type 2 (panel D), PMCA type 1 (panel E), and PMCA type 4 (panel F) are shown. A negative control (primary antibody omitted) is shown in the insert of panel A. In order to outline liver cells, rhodamine-conjugated phalloidin was used to detect actin associated to membrane as showed in the central images. Each figure shows semi-quantitative analysis of calcium-handling protein distribution (pericentral/periportal ratio) for each time (AL-ZT3, AL-ZT6, RF-ZT3, and RF-ZT6) and feeding condition (Fasted and Refed). * (p < 0.05) significant difference between AL time points (ZT3 vs ZT6); # (p < 0.05) AL vs RF; ♦ (p < 0.05) Fasted vs Refed groups and & (p < 0.05) Refed vs RF-ZT6 (Student´s t-test).
Figure 7
Acrophase summary of hepatic calcium-handling protein daily rhythms. Times of peak activity and protein levels for each of the studied proteins are shown for the AL group (black arrowheads) and for the RF group (white arrowheads). The dark gray rectangle indicates the dark period, and the ZT time is shown above. Each arrowhead from the RF group was normalized with respect to its control, and its size indicates the amplitude of the daily rhythm in comparison to the AL group. The dashed rectangle shows the time of food access for the RF group. Calcium-handling protein activity or expression levels that did not show 24-h rhythmicity were omitted.