Daytime food restriction alters liver glycogen, triacylglycerols, and cell size. A histochemical, morphometric, and ultrastructural study

Abstract

Background: Temporal restriction of food availability entrains circadian behavioral and physiological rhythms in mammals by resetting peripheral oscillators. This entrainment underlies the activity of a timing system, different from the suprachiasmatic nuclei (SCN), known as the food entrainable oscillator (FEO). So far, the precise anatomical location of the FEO is unknown. The expression of this oscillator is associated with an enhanced arousal prior to the food presentation that is called food anticipatory activity (FAA). We have focused on the study of the role played by the liver as a probable component of the FEO. The aim of this work was to identify metabolic and structural adaptations in the liver during the expression of the FEO, as revealed by histochemical assessment of hepatic glycogen and triacylglycerol contents, morphometry, and ultrastructure in rats under restricted feeding schedules (RFS).

Results: RFS promoted a decrease in the liver/body weight ratio prior to food access, a reduction of hepatic water content, an increase in cross-sectional area of the hepatocytes, a moderate reduction in glycogen content, and a striking decrease in triacylglyceride levels. Although these adaptation effects were also observed when the animal displayed FAA, they were reversed upon feeding. Mitochondria observed by electron microscopy showed a notorious opacity in the hepatocytes from rats during FAA (11:00 h). Twenty four hour fasting rats did not show any of the modifications observed in the animals expressing the FEO.

Conclusions: Our results demonstrate that FEO expression is associated with modified liver handling of glycogen and triacylglycerides accompanied by morphometric and ultrastructural adaptations in the hepatocytes. Because the cellular changes detected in the liver cannot be attributed to a simple alternation between feeding and fasting conditions, they also strengthen the notion that RFS promotes a rheostatic adjustment in liver physiology during FEO expression.

Figure 1

Water content in the liver of rats exposed to a restricted feeding schedule for 3 weeks (food intake from 12:00 to 14:00 h). Experimental group, black box; ad-libitum fed control group, white box; 24-h fasting control group, hatched and gray box. Data were collected before (08:00 h), during (11:00 h), and after food anticipatory activity (14:00 h). Control group with 24-h fasting was processed at 11:00 h. Results are expressed as mean ± SEM of 6 independent determinations. Significant difference between food-restricted and ad-libitum fed groups [*], within the same experimental group at different times [+], and different from 24-h fasting group [×]. Differences derived from Tukey's post hoc test (α = 0.05).

Figure 2

Toluidine blue-stained histological sections of livers of rats exposed to a restricted feeding schedule for 3 weeks (food intake from 12:00 to 14:00 h). Tissue samples from food-restricted and ad-libitum fed rats were collected before (08:00 h), during (11:00 h), and after food anticipatory activity (14:00 h). The control group with 24-h fasting was processed at 11:00 h. Panels A, C, and E, control ad-libitum fed groups; panels B, D, and F, food-restricted groups; panel G, 24-h fasted group. Images in panels A and B were taken at 08:00 h, in panels C, D and G at 11:00 h, and E and F at 14:00 h.

Figure 3

Quantification of the hepatocytes' cross-sectional area of rats exposed to a restricted feeding schedule for 3 weeks (food intake from 12:00 to 14:00 h). Data are derived from evaluation of the hepatocyte morphology (Figure 2). RFS group, black box; ad-libitum-fed control group, white box; 24-h-fasting control group, hatched and gray box. Results are expressed as mean ± SEM of 6 independent determinations. Significant difference between food restricted and ad-libitum fed groups [*], within the same experimental group [+], and different from 24-h fasting group [×]. Differences derived from Tukey's post hoc test (α = 0.05).

Figure 4

Periodic-acid Schiff (PAS) stained histological sections of livers of rats exposed to a restricted feeding schedule for 3 weeks (food intake from 12:00 to 14:00 h). Pink color indicates the presence of hepatic glycogen. Tissue samples from food-restricted and ad-libitum fed rats were collected before (08:00 h), during (11:00 h), and after food anticipatory activity (14:00 h). The control group with 24-h fasting was processed at 11:00 h. Panels A, C, and E, control ad-libitum fed groups; panels B, D, and F, food-restricted groups; panel G, 24-h fasted group. Images in panels A and B were taken at 08:00 h, in panels C, D and G at 11:00 h, and E and F at 14:00 h.

Figure 5

Quantification of the hepatocytes' glycogen content of rats exposed to a restricted feeding schedule for 3 weeks (food intake from 12:00 to 14:00 h). Data are derived from evaluation of the liver PAS staining from Figure 4. RFS group, black box; ad-libitum-fed control group, white box; 24-h-fasting control group, hatched and gray box. Results are expressed as mean ± SEM of 6 independent determinations. Significant difference between food restricted and ad-libitum fed groups [*], within the same experimental group [+], and different from 24-h fasting group [×]. Differences derived from Tukey's post hoc test (α = 0.05).

Figure 6

Oil red O (ORO)-stained histological sections of livers of rats exposed to a restricted feeding schedule for 3 weeks (food intake from 12:00 to 14:00 h). Intense red color indicates the presence of neutral lipids, mainly triacylglycerols. Tissue samples from food restricted and ad-libitum fed rats were collected before (08:00 h), during (11:00 h), and after food anticipatory activity (14:00 h). Control group with 24-h fasting was processed at 11:00 h. Panels A, C, and E, control ad-libitum fed groups; panels B, D, and F, food-restricted groups; panel G, 24-h fasted group. Images in panels A and B were taken at 08:00 h, in panels C, D and G at 11:00 h, and E and F at 14:00 h.

Figure 7

Quantification of the hepatocytes' triacylglycerols content of rats exposed to a restricted feeding schedule for 3 weeks (food intake from 12:00 to 14:00 h). Data are derived from evaluation of the liver oil red O staining from Figure 6. RFS group, black box; ad-libitum-fed control group, white box; 24-h-fasting control group, hatched and gray box. Results are expressed as mean ± SEM of 6 independent determinations. Significant difference between food restricted and ad-libitum fed groups [*], within the same experimental group [+], and different from 24-h fasting group [×]. Differences derived from Tukey's post hoc test (α = 0.05).

Figure 8

Electron micrographs illustrating liver cells from control (A and D) fasten (B and D) and fed restricted (C and E) rats. Notice that hepatocytes from the fed restricted animal (F) exhibit electron-dense mitochondria (m) surrounded by abundant smooth endoplasmic reticulum (SER). N = cell nucleus, gl = glycogen, asterisks = lipid droplets, arrows = bile canaliculi. Lead-uranium staining. Scale bars = 2 μm in A-C; 0.2 μm in D-E. Representative images of 6 independent experimental observations.

Figure 9

Time of treatment, feeding conditions, times of sampling and light - darkness cycle used in the experimental protocol. RFS = restricted feeding schedule.