Grape-Seed Proanthocyanidin Extract (GSPE) Modulates Diurnal Rhythms of Hepatic Metabolic Genes and Metabolites, and Reduces Lipid Deposition in Cafeteria-Fed Rats in a Time-of-Day-Dependent Manner

Abstract

Scope: Metabolic dysfunction-associated steatotic liver disease (MASLD) is a global health issue with increasing prevalence. Polyphenols, such as grape seed proanthocyanidin extract (GSPE), are bioactive compounds present in plants and represent an interesting therapeutical approach for MASLD.

Methods and results: This study questioned whether the timing of GSPE administration impacts liver diurnal metabolism and steatosis in a rat obesity model. Results from hepatic lipid profiling and diurnal metabolic gene expression and metabolomics reveal that rats fed with a cafeteria (CAF) diet show impaired glucose homeostasis and enhanced lipogenesis in the liver, contributing to liver steatosis. Chronic consumption of GSPE in the inactive or active phase is associated with beneficial effects as the restoration of rhythms of transcripts and metabolites is observed. However, only when given in the active phase, GSPE treatment decreases hepatic triglyceride levels. Using an in vitro hepatocyte model, the study identifies that catechin, one of the main phenolic compounds found in the GSPE extract, is a potential mediator in ameliorating the effects of CAF-induced liver steatosis.

Conclusion: Taken altogether, the findings show that the beneficial effects of GSPE on MASLD development depend on the treatment time.

Keywords: NAFLD/MASLD; chronobiology; circadian rhythms; liver metabolism; obesity; proanthocyanidins.

FIGURE 1

Representative pictures of H&E‐stained liver sections. Scale bar, 100 µm. Rats were fed a STD or CAF diet and received a daily dosage of vehicle or GSPE at the beginning of the light phase (ZT0) (A) or at the beginning of the dark phase (ZT12) (B). Quantitative analysis of lipid droplets in liver samples per microscopic field (100×) (C). * Indicates significant differences (Student's t‐test, *p < 0.05, **p < 0.01, ***p < 0.001). Shown are means ± SEM (n = 16/group).

FIGURE 2

Diurnal rhythms of liver lipid levels and liver weight. Rats were fed STD or CAF diet and received a daily dosage of vehicle or GSPE at the beginning of the light phase (ZT0) (A–D) or at the beginning of the dark phase (ZT12) (E–H). Rhythm parameter determination and comparison were performed using CircaCompare. R/NR indicates significant/non‐significant rhythmicity (p < 0.05). R* shows a tendency of rhythmicity (p < 0.1). N = 3 – 4/group at each time point.

FIGURE 3

Diurnal gene expression profiles of key genes involved in lipid and bile acid metabolism. Rats were fed a STD or CAF diet and received a daily dosage of vehicle or GSPE at the rest phase (ZT0) (A–G) or at the resting phase (ZT12) (H–N). Rhythm parameter determination and comparison were performed using CircaCompare. R/NR indicates significant/non‐significant rhythmicity (p < 0.05). R* shows a tendency of rhythmicity (p < 0.1). N = 3–4/group at each time point.

FIGURE 4

Diurnal gene expression profiles of key genes glucose metabolism. Rats were fed a STD or CAF diet and received a daily dosage of vehicle or GSPE at the rest phase (ZT0) (A–G) or at the resting phase (ZT12) (H–N). Rhythm parameter determination and comparison were performed using CircaCompare. R/NR indicates significant/non‐significant rhythmicity (p < 0.05). R* shows a tendency of rhythmicity (p < 0.1). N = 4/group at each time point.

FIGURE 5

Diurnal hepatic metabolites affected by CAF diet and GSPE treatment given during the inactive or active phase (A–B). Selected metabolites with rhythm parameter alterations in rats treated during the rest or active phase. Rhythm parameter determination and comparison were performed using CircaCompare. R/NR indicates significant/non‐significant rhythmicity (p < 0.05). R* shows a tendency of rhythmicity (p < 0.1). Shown are means ± SEM (n = 4/group at each time point).

FIGURE 6

Effects of palmitate/catechin or epicatechin treatment on circadian clock rhythms in hepatocytes in vitro. A) Luminescence rhythms in synchronized AML12 hepatocytes stably expressing Bmal1‐luc reporter and after treatment with GSPE. Representative dampened sine curve fits on normalized bioluminescence data are shown. B) Circadian rhythm parameters of AML12 Bmal1‐luc cells (amplitude, acrophase, period, and dampening) in response to palmitate/GSPE treatment. C) Gene expression levels of metabolic genes in AML12/Bmal1‐luc cells treated with catechin or epicatechin (10 µM or 100 µM) for 96 h and cultured with bovine serum albumin (BSA) conjugated with palmitate (PAL) (0.25 mM) with 0.1% v/v DMSO. Cells cultured in BSA (0.04 mM) with 0.1% v/v DMSO were used as control. Data are means ± SEM (n = 4). One‐way ANOVA followed by Tukey's post‐test was performed to compare values between groups (*p < 0.05, **p < 0.01, ***p < 0.001).