Long-term restricted feeding alters circadian expression and reduces the level of inflammatory and disease markers

Abstract

The circadian clock in peripheral tissues can be entrained by restricted feeding (RF), a regimen that restricts the duration of food availability with no calorie restriction (CR). However, it is not known whether RF can delay the occurrence of age-associated changes similar to CR. We measured circadian expression of clock genes, disease marker genes, metabolic factors and inflammatory and allergy markers in mouse serum, liver, jejunum and white adipose tissue (WAT) after long-term RF of 4 months. We found that circadian rhythmicity is more robust and is phase advanced in most of the genes and proteins tested under RF. In addition, average daily levels of some disease and inflammatory markers were reduced under RF, including liver Il-6 mRNA, tumour necrosis factor (TNF)-α and nuclear factor κB (NF-κB) protein; jejunum Arginase, Afp, Gadd45β, Il-1α and Il-1β mRNA, and interleukin (IL)-6 and TNF-α protein and WAT Il-6, Il-1β, Tnfα and Nfκb mRNA. In contrast, the anti-inflammatory cytokine Il-10 mRNA increased in the liver and jejunum. Our results suggest that RF may share some benefits with those of CR. As RF is a less harsh regimen to follow than CR, the data suggest it could be proposed for individuals seeking to improve their health.

Fig 1

Body weight and locomotor activity of mice fed AL or RF. (A) Mean body weight during AL and RF. After 2 weeks of AL food availability, food was given either AL or restricted in time for only 3 hrs between ZT3 and ZT6. Body weight of mice fed AL (solid black line) or RF (dashed grey line) diet was measured at the end of each week throughout the experiment. Values are means ± S.E., n = 4 for each time-point in each group. (B) Representative double-plotted actograms of AL- and RF-fed mice. The white and black bars designate the light and dark periods, respectively. Food availability during RF is marked by the crosshatched box. DD indicates the day on which mice were put in total darkness. (C) Animal locomotor activity of mice fed AL (solid black line) or RF (dashed grey line) during the first day in DD. The grey and black bars designate the subjective day and night, respectively. Food availability during RF is marked by the crosshatched box.

Fig 2

Circadian rhythms of clock genes in the liver and jejunum of AL- and RF-fed mice. Liver and jejunum were collected every 3 hrs around the circadian cycle from mice fed either AL (solid black line) or RF (dashed grey line). Food availability during RF is marked by the crosshatched box. mRNA was quantified by real-time PCR. Clock gene levels were normalized using Gapdh as the reference gene. The grey and black bars designate the subjective day and night, respectively. Values are means ± S.E., n = 4 for each time-point in each group.

Fig 3

Circadian rhythms and average mRNA levels of metabolic markers in the liver, jejunum and WAT of AL- and RF-fed mice. Liver and jejunum were collected every 3 hrs around the circadian cycle from mice fed either AL (solid black line and columns) or RF (dashed grey line and grey columns). Food availability during RF is marked by the crosshatched box. mRNA was quantified by real-time PCR and is plotted as relative levels. Metabolic gene levels were normalized using Gapdh as the reference gene. For total daily levels, all time-points were averaged. The grey and black bars designate the subjective day and night, respectively. Values are means ± S.E., n = 4 for each time-point in each group. Asterisk denotes significant difference (P < 0.05).

Fig 4

Circadian rhythms and average protein levels of AMPK, pAMPK and SIRT1 in the liver and jejunum of AL- and RF-fed mice. Liver and jejunum were collected every 3 hrs around the circadian cycle from mice fed either AL (solid black line and columns) or RF (dashed grey line and grey columns). Food availability during RF is marked by the crosshatched box. Protein was analysed by Western blotting and quantified using actin as loading control. Representative blots are shown above the figures. For total daily levels, all time-points were averaged. The grey and black bars designate the subjective day and night, respectively. Values are means ± S.E., n = 4 for each time-point in each group. Asterisk denotes significant difference (P < 0.05).

Fig 5

Serum levels of lipids, inflammation and disease markers of AL- and RF-fed mice. Blood was collected and serum separated for analysis every 3 hrs around the circadian cycle (triglycerides, cholesterol, IL-6, ALT and AST) or at mid-day and mid-night (CRP, PAI-1) from mice fed either AL (solid black line and columns) or RF (dashed grey line and grey columns). For triglycerides, food availability during RF is marked by the crosshatched box. Protein and lipid levels were determined by ELISA. For total daily levels, all time-points were averaged. The grey and black bars designate the subjective day and night, respectively. Values are means ± S.E., n = 3 for each time-point in each group. Asterisk denotes significant difference (P < 0.05).

Fig 6

Average mRNA levels of disease markers in the liver and jejunum of AL- and RF-fed mice. Liver and jejunum were collected every 3 hrs around the circadian cycle from mice fed either AL (black columns) or RF (grey columns). mRNA was quantified by real-time PCR. Disease marker gene levels were normalized using Gapdh as the reference gene. For total daily levels, all time-points were averaged. Values are means ± S.E., n = 48 for each group. Asterisk denotes significant difference (P < 0.05).

Fig 7

Circadian rhythms and average mRNA levels of inflammation markers in the liver, jejunum and WAT of AL- and RF-fed mice. Liver, jejunum and WAT were collected every 3 hrs around the circadian cycle from mice fed either AL (solid black line and columns) or RF (dashed grey lined and grey columns). Food availability during RF is marked by the crosshatched box. mRNA was quantified by real-time PCR. Inflammation gene levels were normalized using Gapdh as the reference gene. For total daily levels, all time-points were averaged. The grey and black bars designate the subjective day and night, respectively. Values are means ± S.E., n = 6 for each time-point in each group. Asterisk denotes significant difference (P < 0.05).

Fig 8

Circadian rhythms and average protein levels of inflammation markers in the liver and jejunum of AL- and RF-fed mice. Liver and jejunum were collected every 3 hrs around the circadian cycle from mice fed either AL (solid black line and columns) or RF (dashed grey line and grey columns). Food availability during RF is marked by the crosshatched box. Protein was analysed by Western blotting and quantified using actin as loading control. For total daily levels, all time-points were averaged. The grey and black bars designate the subjective day and night, respectively. Values are means ± S.E., n = 3 for each time-point in each group. Asterisk denotes significant difference (P < 0.05).

Fig 9

Circadian rhythms and average mRNA levels of mast cell markers in the jejunum of AL- and RF-fed mice. Jejunum was collected every 3 hrs around the circadian cycle from mice fed either AL (solid black line and columns) or RF (dashed grey lined and grey columns). Food availability during RF is marked by the crosshatched box. mRNA was quantified by real-time PCR. Gene levels were normalized using Gapdh as the reference gene. For total daily levels, all time-points were averaged. The grey and black bars designate the subjective day and night, respectively. Values are means ± S.E., n = 6 for each time-point in each group. Asterisk denotes significant difference (P < 0.05).

Fig 10

Suggested model for RF effects and interactions in various mouse tissues. (A) In the liver, RF is believed to lead to an increase in both NAD⁺ and AMP levels that could explain the observed AMPK activity up-regulation and increased Sirt1 mRNA leading to presumed increased activity levels. AMPK phosphorylates PGC-1α and the observed increase in its mRNA levels alongside those of Sirt1, assuming a parallel increase at the protein level, may lead to the arrest of glycolysis and fat storage and increase in gluconeogenesis and fatty acid oxidation. SIRT1 also inhibits NF-κB activity, as observed, which leads to down-regulation of pro-inflammatory cytokines (IL-6 and TNF-α). Combined with the observed up-regulation of the anti-inflammatory cytokine Il-10 mRNA, assuming similar effect at the protein level, this yields reduced inflammation. (B) In the jejunum, RF increases Pparα mRNA levels, which leads to fatty acid oxidation, assuming a parallel increase at the protein level. PPAR-α is also known to inhibit NF-κB. Pro-inflammatory cytokines (IL-1, IL-6 and TNF-α) are down-regulated and together with an increase of the anti-inflammatory cytokine IL-10 ultimately lead to reduced inflammation. This suggests a novel pathway by which RF can influence the inflammatory processes in the gut. (C) In WAT, inflammatory processes are inhibited in fat tissue as well. In addition, RF decreases Pparγ mRNA and assuming a parallel decrease at the protein level, this could result in reduced fat storage.