Rhythm and ROS: Hepatic Chronotherapeutic Features of Grape Seed Proanthocyanidin Extract Treatment in Cafeteria Diet-Fed Rats

Abstract

Polyphenols play a key role in the modulation of circadian rhythms, while the cafeteria diet (CAF) is able to perturb the hepatic biological rhythm and induce important ROS production. Consequently, we aimed to elucidate whether grape seed proanthocyanidin extract (GSPE) administration recovers the CAF-induced hepatic antioxidant (AOX) misalignment and characterize the chronotherapeutic properties of GSPE. For this purpose, Fischer 344 rats were fed a standard diet (STD) or a CAF and concomitantly treated with GSPE at two time-points (ZT0 vs. ZT12). Animals were euthanized every 6 h and the diurnal rhythms of hepatic ROS-related biomarkers, hepatic metabolites, and AOX gene expression were examined. Interestingly, GSPE treatment was able to recover the diurnal rhythm lost due to the CAF. Moreover, GSPE treatment also increased the acrophase of Sod1, as well as bringing the peak closer to that of the STD group. GSPE also corrected some hepatic metabolites altered by the CAF. Importantly, the differences observed at ZT0 vs. ZT12 due to the time of GSPE administration highlight a chronotherapeutic profile on the proanthocyanin effect. Finally, GSPE could also reduce diet-induced hepatic oxidative stress not only by its ROS-scavenging properties but also by retraining the circadian rhythm of AOX enzymes.

Keywords: GSPE; cafeteria diet; chronotherapy; circadian rhythms; diurnal rhythms; liver; oxidative stress; phenolic compounds; proanthocyanidins; zeitgeber.

Figure 1

Experimental design. Ninety-six Fischer 344 rats were randomly divided into two groups based on their diet: 32 were fed STD and 64 were fed CAF for 5 weeks. Then, each diet group was further divided into two groups for STD-fed rats and four groups for CAF-fed rats, to receive either VH or GSPE doses. Both STD and CAF-fed rats were administered VH at two different time points: 8 a.m. (ZT0) and 8 p.m. (ZT12), while CAF-fed rats were additionally given GSPE (25 mg/kg b.w.) at the same time points. Finally, each experimental group was subdivided into four based on the time of death of the animals: 9 a.m. (ZT1), 3 p.m. (ZT7), 9 p.m. (ZT13), and 3 a.m. (ZT19).

Figure 2

Body weight gain (% to Week 0). (A) Body weight gain was expressed in the percentage of ZT0-treated rats (STD-VH, CAF-VH, and CAF-GSPE) during the nine weeks of the experiment. (B) Body weight gain was expressed in the percentage of ZT12-treated rats (STD-VH, CAF-VH, and CAF-GSPE) during the nine weeks of the experiment. Data are shown as mean ± S.D (n = 14–16). $ indicates significant differences by Student’s t-test between STD-VH vs. CAF-VH (p < 0.001). # indicates significant differences by Student’s t-test between CAF-VH vs. CAF-GSPE (p < 0.001). t, indicates time effect; D, diet effect, D*t, interaction between diet and time, T, indicates treatment effect, and T*t, interaction between treatment and time via 2-way ANOVA. (C) AUC of the percentage of the BWG from week 5. Data are shown as mean ± S.E.M (n = 14–16). + indicates significant differences by Student’s t-test between STD-VH vs. CAF-VH (p < 0.05). * Indicates significant differences by Student’s t-test between CAF-VH vs. CAF-GSPE (p < 0.05). STD, rats fed a Standard diet; CAF, rats fed a Cafeteria diet; VH, rats administered vehicle; GSPE, rats administered 25 mg/kg b.w. grape seed proanthocyanidin extract.

Figure 3

Estimated diurnal rhythms of liver SOD (A), Catalase (B), GPx1 (C), GSH (D), and Thiols (E) in ZT0-treated rats. Data are shown as the median ± Min to max, its diurnal oscillation, and acrophases with their amplitude. + indicates significant differences (p < 0.05) by diet effect (STD-VH vs. CAF-VH), $ indicates tendency (0.1 > p ≥ 0.05) by diet effect; # indicates tendency (0.1 > p ≥ 0.05) by treatment effect (CAF-VH vs. CAF-GSPE) using Mann–Whitney U test. STD, rats fed a Standard diet; CAF, rats fed a Cafeteria diet; VH, rats administered vehicle; GSPE, rats administered 25 mg/kg b.w. grape seed proanthocyanidin extract; Arrow, time of treatment administration.

Figure 4

Estimated diurnal rhythms of liver SOD (A), Catalase (B), GPx1 (C), GSH (D), and Thiols (E) in ZT12-treated rats. Data are shown as the median ± Min to max, its diurnal oscillation, and acrophases with their amplitude. + indicates significant differences (p < 0.05) by diet effect (STD-VH vs. CAF-VH), $ indicates tendency (0.1 > p ≥ 0.05) by diet effect using Mann–Whitney U test. STD, rats fed a Standard diet; CAF, rats fed a Cafeteria diet; VH, rats administered vehicle; GSPE, rats administered 25 mg/kg b.w. grape seed proanthocyanidin extract; Arrow, time of treatment administration.

Figure 5

Estimated diurnal rhythms of liver Sod1 (A), Sod2 (B), Catalase (C), GPx1 (D), and GSR (E) gene expression in ZT0-treated rats. Data are shown as the median ± Min to max, its diurnal oscillation, and acrophases with their amplitude. $ indicates tendency (0.1 > p ≥ 0.05) by diet effect (STD-VH vs. CAF-VH); * indicates significant differences (p < 0.05) by treatment effect (CAF-VH vs. CAF-GSPE) using Mann-Whitney U test. STD, rats fed a Standard diet; CAF, rats fed a Cafeteria diet; VH, rats administered vehicle; GSPE, rats administered 25 mg/kg b.w. grape seed proanthocyanidin extract; Arrow, time of treatment administration.

Figure 6

Estimated diurnal rhythms of liver Sod1 (A), Sod2 (B), Catalase (C), GPx1 (D), and GSR (E) gene expression in ZT12-treated rats. Data are shown as the median ± Min to max, its diurnal oscillation, and acrophases with their amplitude. $ indicates tendency (0.1 > p ≥ 0.05) by diet effect (STD-VH vs. CAF-VH); * indicates significant differences (p < 0.05) by treatment effect (CAF-VH vs. CAF-GSPE), # indicates tendency (0.1 > p ≥ 0.05) by treatment effect using Mann–Whitney U test. STD, rats fed a Standard diet; CAF, rats fed a Cafeteria diet; VH, rats administered vehicle; GSPE, rats administered 25 mg/kg b.w. grape seed proanthocyanidin extract; Arrow, time of treatment administration.

Figure 7

Hepatic levels of (A) α-tocopherol, (B) citric acid, (C) taurine, (D) glycine, and (E) glutamic acid of rats treated at ZT0. Data are shown as the median ± Min to max (n = 4). + indicates significant differences (p < 0.05) by diet effect (STD-VH vs. CAF-VH), $ indicates tendency (0.1 > p ≥ 0.05) by diet effect; # indicates tendency (0.1 > p ≥ 0.05) by treatment effect (CAF-VH vs. CAF-GSPE) using Mann–Whitney U test. STD, rats fed a Standard diet; CAF, rats fed a Cafeteria diet; VH, rats administered vehicle; GSPE, rats administered 25 mg/kg b.w. grape seed proanthocyanidin extract, A.U., arbitrary units.

Figure 8

Hepatic levels of (A) α-tocopherol, (B) citric acid, (C) taurine, (D) glycine, and (E) glutamic acid of rats treated at ZT12. Data are shown as the median ± Min to max (n = 4). + indicates significant differences (p < 0.05) by diet effect (STD-VH vs. CAF-VH), $ indicates tendency (0.1 > p ≥ 0.05) by diet effect; * indicates significant differences (p < 0.05) by treatment effect (CAF-VH vs. CAF-GSPE) using Mann–Whitney U test. STD, rats fed a Standard diet; CAF, rats fed a Cafeteria diet; VH, rats administered vehicle; GSPE, rats administered 25 mg/kg b.w. grape seed proanthocyanidin extract, A.U., arbitrary units.

Figure 9

Potential mechanisms of action in which the chronotherapeutic effects of GSPE could be involved in the liver of diet-induced obese rats. The CAF induces an oxidative imbalance, thus enhancing ROS production, which leads to the loss of diurnal rhythmicity in certain AOX-related parameters such as GPx1, GSH, or thiols. However, oral GSPE intake has an influence on BMAL1 throughout its acetylation during the activity phase (dark phase in rats), contributing to restoring circadian rhythmicity in clock-controlled genes such as Sod1 or Nrf2. The improvement in Nrf2 expression mediated by BMAL1 contributes to alleviating the redox imbalance, reducing ROS, and achieving restoration of the diurnal rhythmicity of GPx1, GSH, and thiols. However, GSPE could also act as a chronotherapy, decreasing the body weight gain when administered at ZT12 (beginning of the active phase) but not when administered at ZT0 (resting phase), which could be mediated by the differential bioavailability of the extract depending on the time of administration and the differential day–night dynamics of the gut microbiota, which also influences GSPE bioavailability. BMAL1: Brain and muscle ARNT-Like 1; CLOCK: Circadian locomotor output cycles kaput; SIRT1: Sirtuin-1; Per: Period; Cry: Cryptochrome; Rorα: RAR-related orphan receptor alpha; Rev-erbα: Rev-Erb alpha; CCGs: Clock-controlled genes; NRF2: Nuclear factor erythroid 2-related factor 2; GSPE: Grape seed proanthocyanidin extract; AOX: Antioxidant; ROS: Reactive oxygen species; GPx1: Glutathione peroxidase 1; GSH: Glutathione; ZT: Zeitgeber. Arrowed lines: activation or induction; lines without an arrow: inhibition or negative regulation.