Proanthocyanidins Restore the Metabolic Diurnal Rhythm of Subcutaneous White Adipose Tissue According to Time-Of-Day Consumption

Abstract

Consumption of grape seed proanthocyanidin extract (GSPE) has beneficial effects on the functionality of white adipose tissue (WAT). However, although WAT metabolism shows a clear diurnal rhythm, whether GSPE consumption could affect WAT rhythmicity in a time-dependent manner has not been studied. Ninety-six male Fischer rats were fed standard (STD, two groups) or cafeteria (CAF, four groups) diet for 9 weeks (n = 16 each group). From week 6 on, CAF diet animals were supplemented with vehicle or 25 mg GSPE/kg of body weight either at the beginning of the light/rest phase (ZT0) or at the beginning of the dark/active phase (ZT12). The two STD groups were also supplemented with vehicle at ZT0 or ZT12. In week 9, animals were sacrificed at 6 h intervals (n = 4) to analyze the diurnal rhythms of subcutaneous WAT metabolites by nuclear magnetic resonance spectrometry. A total of 45 metabolites were detected, 19 of which presented diurnal rhythms in the STD groups. Although most metabolites became arrhythmic under CAF diet, GSPE consumption at ZT12, but not at ZT0, restored the rhythmicity of 12 metabolites including compounds involved in alanine, aspartate, and glutamate metabolism. These results demonstrate that timed GSPE supplementation may restore, at least partially, the functional dynamics of WAT when it is consumed at the beginning of the active phase. This study opens an innovative strategy for time-dependent polyphenol treatment in obesity and metabolic diseases.

Keywords: acrophase; chronobiology; chrononutrition; circacompare; flavonoids; metabolomics; zeitgeber.

Figure 1

Overlap diagram of rhythmic metabolite concentrations presented in ZT0 (a) and ZT12 groups (b). Rats were fed standard or cafeteria diet for 9 weeks. At week 6, rats received a daily oral dose of GSPE or vehicle for 4 weeks at ZT0 or ZT12. Rats from each group were sacrificed at four different time points: ZT1, ZT7, ZT13, and ZT19 in order to analyze the diurnal rhythm of these metabolites. Forty-five metabolites detected by NMR were analyzed using CircaCompare algorithm based on Cosinor (p < 0.05). Metabolites from aqueous phase are shown in black color and metabolites from lipidic phase in brown color. Abbreviations: iWAT, inguinal adipose tissue; ZT0, beginning of the light phase; ZT12, beginning of the dark phase; STD-VH-ZT0; standard diet vehicle group treated at ZT0; CAF-VH-ZT0, cafeteria diet vehicle group treated at ZT0; CAF-GSPE-ZT0, cafeteria diet treated with grape-seed procyanidin extract at ZT0; STD-VH-ZT12; standard diet vehicle group treated at ZT12; CAF-VH-ZT12, cafeteria diet vehicle group treated at ZT12; CAF-GSPE-ZT12, cafeteria diet treated with grape seed procyanidin extract at ZT12; 3-OHB, 3-Hydroxybutyrate; Chol, cholesterol; Total FA, total fatty acids; TAG, triacylglyceride; MUFA, monounsaturated fatty acids; ω-3, omega-3; GPC, glycerophosphocholine; Phe, phenylalanine; ChoP, phosphorylcholine.

Figure 2

Lipophilic metabolites that recovered their diurnal rhythm loss under cafeteria diet in response to GSPE administration at ZT12. Circadian parameters such as rhythmicity, mesor, amplitude, and acrophase were calculated using the CircaCompare algorithm based on Cosinor. Graphs showing diurnal rhythm and acrophase were performed using the Python package based on Cosinor. R indicates significant rhythmicity; NR indicates non-rhythmic; M indicates significant mesor difference between STD-VH-ZT12 and both cafeteria diet groups; * indicates significant acrophase difference between STD-VH-ZT vs CAF-GSPE-ZT12. None of the metabolites presented differences between groups for amplitude.

Figure 3

Hydrophilic metabolites which recovered their diurnal rhythm lost under cafeteria diet through GSPE administration in ZT12. Circadian parameters such as rhythmicity, mesor, amplitude, and acrophase were calculated using the CircaCompare algorithm based on Cosinor. Graphs showing diurnal rhythm and acrophase were performed using the Python package based on Cosinor. R, indicates significant rhythmicity; NR, indicates non-rhythmic; M, denotes significant mesor difference against STD-VH-ZT12; A, denotes significant amplitude difference against STD-VH-ZT12; * indicates significant acrophase difference between STD-VH-ZT vs CAF-GSPE-ZT12.

Figure 4

Metabolic network analysis of identified rhythmic metabolites in iWAT. Hydrophilic metabolites with diurnal rhythm in the corresponding groups, (a) inter-tissue amino acid flux and alanine, aspartate, and glutamate metabolism pathway (b). Green boxes indicate metabolites involved in alanine, aspartate, and glutamate metabolism that are rhythmic in STD-VH-ZT12, CAF-GSPE-ZT12, or both groups; yellow boxes indicate metabolites that are rhythmic in both STD-VH-ZT12 and CAF-GSPE-ZT12 that are not involved in alanine, aspartate, and glutamate metabolism. Abbreviations: ChoP, phosphorylcholine; GPC, glycerophosphocholine; Phe, phenylalanine; TAG, triacylglyceride; Glycerol 3-P, glycerol 3-phosphate.