Grape Seed Flavanols Restore Peripheral Clock of White Adipose Tissue in Obese Rats Under Circadian Alterations

Abstract

Background: White adipose tissue (WAT) exhibits diurnal oscillations regulated by clock genes, which autonomously control its functionality. These rhythms are modulated by the central clock and external factors, such as light exposure and diet. Flavanols, phenolic compounds known for their beneficial metabolic effects, have been shown to modulate the expression of clock genes. This study explored the impact of flavanols on clock gene expression in WAT explants from lean and obese rats under changes in light/dark cycles. Methods: WAT explants were obtained from 24 Fischer rats fed a standard diet (STD) or cafeteria diet (CAF) for seven weeks. During the final week, rats were changed to short (6 h of light, L6) or long (18 h of light, L18) photoperiods. CAF-fed rats were also administered a grape seed (poly)phenol-rich extract (GSPE) (25 mg/kg) or vehicle (VH). After sacrifice, WAT explants were collected every 6 h starting at 8 a.m. the following day (CT0, CT6, CT12, CT18, and CT24). Results: The results showed that under L18 conditions, STD-fed rats displayed oscillations in Bmal1, Cry1, Per1, and Rev-erbα clock gene expression, whereas many of these rhythms were disrupted under L6 conditions. Moreover, the administration of the CAF diet also resulted in the loss of clock gene circadian oscillations in the WAT explants. GSPE administration restored the oscillation of these clock genes under L18 and L6 conditions. Conclusions: These findings highlight the potential zeitgeber role of flavanols in modulating WAT peripheral clocks and their capacity to improve metabolic and circadian regulation under conditions of diet- and photoperiod-induced disruption.

Keywords: chrononutrition; circadian rhythms; metabolism; phenolic compounds; photoperiod; zeitgeber.

Figure 1

(A) Final body weight and (B) body weight gain during the last week of the experiment for L6 and L18 conditions (STD-VH, CAF-VH and CAF-GSPE) in the seventh week. D, diet effect; T, GSPE administration effect. * or ** indicate significant differences between groups (p ≤ 0.05 and p ≤ 0.01, respectively) and # indicates tendency (p = 0.1–0.051) using 2-way ANOVA followed by LSD post hoc test. STD, standard diet-fed rats; CAF, cafeteria diet-fed rats; VH, rats administered vehicle; GSPE, rats administered 25 mg/kg grape seed (poly)phenol extract; L6, short photoperiod, 6 h light per day; L18, long photoperiod, 18 h light per day.

Figure 2

Relative gene expression of core clock genes in inguinal white adipose tissue (iWAT). Bmal1, Cry1, and Per1 relative gene expressions in iWAT under L6 (A,C,E) and L18 (B,D,F) conditions. * indicates significant differences (p < 0.05) and # tendency (p = 0.1–0.051) between groups at each time point by Mann–Whitney test. Significant differences or trends between groups by photoperiod are indicated in the L6 graphs above the T symbol. STD, standard diet-fed rats; CAF, cafeteria diet-fed rats; VH, rats administered vehicle; GSPE, rats administered 25 mg/kg grape seed (poly)phenol extract; L6, short photoperiod, 6 h light per day; L18, long photoperiod, 18 h light per day; CT, circadian time.

Figure 3

Estimated circadian oscillations of core clock genes in inguinal white adipose tissue. Estimated circadian rhythms and acrophases (indicated by the arrows) with their amplitude (indicated by the arrow size) represent Bmal1 genes for STD-VH, CAF-VH, and CAF-GSPE under (A) L6 and (B) L18 conditions. Estimated circadian rhythms and acrophases with their amplitude represent Cry1 genes for STD-VH, CAF-VH, and CAF-GSPE under (C) L6 and (D) L18 conditions. Estimated circadian rhythms and acrophases with their amplitude represent Per1 genes for STD-VH, CAF-VH, and CAF-GSPE under (E) L6 and (F) L18 conditions. STD, standard diet-fed rats; CAF, cafeteria diet-fed rats; VH, rats administered vehicle; GSPE, rats administered 25 mg/kg grape seed (poly)phenol extract; L6, short photoperiod, 6 h light per day; L18, long photoperiod, 18 h light per day; CT, circadian time; R, significant or tends to rhythmic; NR, non-rhythmic.

Figure 4

Relative gene expression of clock-controlled genes in inguinal white adipose tissue. Rev-erbα relative gene expression in iWAT under (A) L6 and (B) L18 conditions. Nampt relative gene expression in iWAT under (C) L6 and (D) L18 conditions. * indicates significant differences (p < 0.05) and # indicates the tendency (p = 0.1–0.051) between groups at each time point by Mann–Whitney test. Significant differences or trends between groups by photoperiod are indicated in the L6 graphs above the T symbol. STD, standard diet-fed rats; CAF, cafeteria diet-fed rats; VH, rats administered vehicle; GSPE, rats administered 25 mg/kg grape seed (poly)phenol extract; L6, short photoperiod, 6 h light per day; L18, long photoperiod, 18 h light per day; CT, circadian time.

Figure 5

Estimated circadian oscillations of clock-controlled genes in inguinal white adipose tissue. Estimated circadian rhythms and acrophases (indicated by the arrows) with their amplitude (indicated by the arrow size) representing Rev-erbα genes for STD-VH, CAF-VH, and CAF-GSPE under (A) L6 and (B) L18 conditions. Estimated circadian rhythms and acrophases with their amplitude representing Nampt genes for STD-VH, CAF-VH, and CAF-GSPE under (C) L6 and (D) L18 conditions. STD, standard diet-fed rats; CAF, cafeteria diet-fed rats; VH, rats administered vehicle; GSPE, rats administered 25 mg/kg grape seed (poly)phenol extract; L6, short photoperiod, 6 h light per day; L18, long photoperiod, 18 h light per day; CT, circadian time; R, significant or tends to rhythmic; NR, non-rhythmic.

Figure 6

Summary diagram of the relationship between clock genes and observed effects. The proposed pathways that are altered by the CAF diet and that could promote detrimental effects on the peripheral clock of iWAT and metabolism, depending on the photoperiod to which the animals were exposed. In addition, the possible clock genes involved in the beneficial effects of GSPE on the damage caused by the CAF diet are shown. Under L6 conditions, lower insulin sensitivity was observed in obese rats and iWAT explants showed a loss of circadian oscillation in Bmal1, Per1, Nampt, and Rev-erbα. Obese rats under L18 conditions displayed a worsened serum lipid profile, while iWAT explants from these animals showed circadian disruption in genes such as Bmal1, Cry1, and Nampt. With GSPE supplementation, the animals showed improved insulin sensitivity under L6 conditions, exhibiting circadian rhythmicity in genes such as Bmal1 and Nampt. These genes are closely related to insulin sensitivity [52]. GSPE supplementation under L18 conditions improved cholesterol and triglyceride levels, while their explants showed rhythmicity in clock genes, such as Cry1 and Rev-erbα, involved in the regulation of lipid metabolism [53,54,55,56], which could suggest better circadian regulation of pathways such as lipolysis and lipogenesis. CAF, cafeteria diet-fed rats; GSPE, rats administered 25 mg/kg grape seed (poly)phenol extract; L6, short photoperiod, 6 h light per day; L18, long photoperiod, 18 h light per day.