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Grape seed procyanidins improve atherosclerotic risk index and induce liver CYP7A1 and SHP expression in healthy rats

Departament de Bioquímica i Biotecnologia. CeRTA. Universitat Rovira i Virgili, Tarragona, Spain

²Correspondence: Departament de Bioquímica i Biotecnologia, Universitat Rovira i Virgili, Plaça Imperial Tarraco 1, Tarragona 43005, Spain. E-mail: mcbs{at}astor.urv.es

SPECIFIC AIMS

Moderate consumption of red wine reduces risk of death from cardiovascular disease. Red wine polyphenols (RWPs) are ultimately responsible for this effect, exerting antiatherogenic actions through their antioxidant capacities and by modulating intracellular signaling pathways and transcriptional activity. Lipoprotein metabolism is crucial in atherogenesis, and the liver is the principal organ controlling lipoprotein homeostasis. The aim of this study was to investigate short-term effects of procyanidins, the most abundant polyphenols present in red wine, in vivo, and in healthy (normolipidemic) animals in order to gain insight on the primary mechanisms that underlie the long-term antiatherogenic and cardioprotector effects ascribed to RWPs. We orally administered a single high and nontoxic dose (250 mg/kg BW) of grape seed procyanidins extract (GSPE group) to chow-fed male Wistar rats weighing 250 g and analyzed the plasma lipid and lipoprotein profile after 5 h. Changes in the gene expression pattern in the liver of GSPE-treated animals were analyzed using microarray hybridizations to identify procyanidins target genes involved in lipid metabolism. Lipoprotein lipase (LPL) mRNA levels in muscle and adipose tissue were analyzed by quantitative RT-PCR. Animals treated under the same conditions, without GSPE addition, were used as the control group.

PRINCIPAL FINDINGS

1. Procyanidins improve postprandial lipemia
Five hours after oral administration of GSPE, LDL-C and nonHDL:nonLDL-C levels were significantly lowered in the GSPE group (50 and 30%, respectively), and HDL-C levels were slightly increased (10%) (Table 1 ). However, no statistical differences in plasma total cholesterol (TC) content were found. HDL-C/LDL-C increased by 2-fold whereas the TC/HDL-C ratio decreased 1.3-fold in GSPE-treated animals. TG and apoB levels in plasma were reduced to 50% and 40% (Fig. 1 ), respectively, indicating that GSPE ingestion affects chylomicron and/or VLDL synthesis and/or utilization by peripheral tissues.

View larger version (31K): [in this window] [in a new window]   Figure 1. Plasma total apolipoprotein B, apolipoprotein B-100 and apolipoprotein B-48 in control rats and grape seed procyanidin extract (GSPE) -treated rats. A) Representative ApoB-48 and ApoB-100 inmunoblot analysis of a control (lanes 1, 2), GSPE (lanes 4, 5) plasma samples, and apoB-100 standard (lane 3). Rats were treated with an oral gavage of GSPE (250 mg/kg BW) and sacrificed after 5 h. Plasma samples (120 µg of protein) and apoB-100 standard (0.2 µg) were subjected to SDS-PAGE and transferred onto PVDF membrane for inmunoblot analysis with goat anti-apoB antibody. The antibody used was raised against the amino terminus of apoB so both isoforms, apoB-100 and apoB-48, could be detected and quantified simultaneously in a single plasma sample. B) Amounts of plasma total-ApoB, ApoB-48 and ApoB-100 in control group and GSPE-treated group. ApoB-100 standard was used to quantify the relative amount of apoB-100 and apoB-48 using Quantity One software from Bio-Rad. Data are expressed as mean ± SE (n=6). *Significant difference at the P<0.05 level vs. control value.

In the liver, levels of total lipids, TG, TC, free cholesterol, and esterified cholesterol were not affected by GSPE treatment.

2. Procyanidins modify liver expression of genes involved in lipid metabolism
Microarray hybridization analysis of transcript profile revealed that GSPE ingestion had relevant effects on liver gene expression. The mRNA level of CYP7A1, the key enzyme controlling bile acid synthesis, was increased 2.4-fold in the GSPE-treated rats. The cholesterol biosynthesis pathway was also up-regulated in the GSPE group: mRNA levels of HMG-CoA reductase, HMG-CoA synthase, squalene epoxidase, and sterol-C4-methyl oxidase increased by > 1.4-fold. No changes were found in the expression of cholesterol estherification, fatty acid synthesis, or fatty acid oxidation enzymes. These results, along with the fact that cholesterol levels in liver and plasma remained unchanged, suggest that cholesterol biosynthesis induced by GSPE is directed to bile acids production.

Concerning apolipoprotein gene expression, ApoC-I, ApoC-III, and ApoC-II decreased their mRNA levels to 0.80, 0.81, and 0.67 of control values, respectively. These changes strongly suggest that GSPE induces qualitative changes in secreted lipoproteins, which could contribute to the TG decrease observed and the modifications in cholesterol distribution in plasma.

Expression of small heterodimer partner 1 (SHP), a nuclear receptor emerging as a key cotranscriptional factor in the control of lipid homeostasis, showed a 3-fold increase in the GSPE-treated group. Mutations in SHP have been associated with mild obesity and insulin resistance. Moreover, other hypolipidemic agents (such as guggulsterone and some estrogens) induce SHP expression. Thus, GSPE could exert its hypolipidemic effects through the up-regulation of SHP transcription.

3. Procyanidins modify lipoprotein lipase (LPL) expression in peripheral tissues
Plasma triglyceride levels markedly depend on their uptake by peripheral tissues, mainly muscle and adipose tissue, controlled by the activity of LPLs. Muscle LPL mRNA was increased 1.57-fold and adipose tissue LPL mRNA was decreased to 0.57-fold of control. These results strongly suggest that plasma TG utilization in GSPE-treated animals is preferentially directed toward energy production by the muscle instead of for energy storage by the adipose tissue.

CONCLUSIONS AND SIGNIFICANCE

Our experimental design is novel in that we assess the short-term effects of grape seed procyanidins ingestion in vivo on healthy animals fed a standard diet. Our results reinforce and could partially explain earlier studies performed in vitro or with dyslipidemic in vivo models using chronic ingestion of grape seed procyanidins.

The relevant novel findings of our study are: 1) GSPE displayed a drastic hypolipidemic and antiatherogenic effect in a postprandial situation, pointing out a protector role of grape seed procyanidins; 2) these effects were accompanied by the induction of CYP7A1 expression in liver, suggesting increased cholesterol elimination via bile acids; 3) GSPE could exert its hypolipidemic effects through the up-regulation of SHP transcription; and 4) lipoprotein lipase is up-regulated in muscle and down-regulated in adipose tissue, suggesting changes in the utilization of TG by the body.

If the improvement noted in lipemia induced by oral administration of procyanidins in rats were functional in humans, in whom postprandial lipemia increases the risk of atherogenesis and coronary artery disease, the consumption of red wine associated with meals could be fundamental to explain the long-term beneficial effects described by the "French Paradox."

View larger version (34K): [in this window] [in a new window]   Figure 2. Schematic representation of grape seed procyanidins effects. Procyanidins and/or their metabolites modify gene expression in liver, muscle, and adipose tissue. In liver, expression of SHP and CYP7A1 is up-regulated whereas ApoA2, ApoC1, and ApoC3 expression is down-regulated. LPL expression is up-regulated in muscle and down-regulated in adipose tissue. These changes could explain the effect of GSPE seen in plasma TG and apoB levels and cholesterol distribution by affecting VLDL synthesis and secretion, reverse transport of cholesterol, and consumption of TG by muscle and adipose tissue. Thus, GSPE ingestion reduces atherosclerotic risk and could redirect triglyceride to energy production (muscle) instead of to energy storage (adipose tissue).

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3095fje;

¹ These authors contributed equally to this work.

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