Chronic consumption of dietary proanthocyanidins modulates peripheral clocks in healthy and obese rats.

Circadian rhythm plays an important role in maintaining homeostasis, and its disruption increases the risk of developing metabolic syndrome. Circadian rhythm is maintained by a central clock in the hypothalamus that is entrained by light, but circadian clocks are also present in peripheral tissues. These peripheral clocks are trained by other cues, such as diet. The aim of this study was to determine whether proanthocyanidins, the most abundant polyphenols in the human diet, modulate the expression of clock and clock-controlled genes in the liver, gut and mesenteric white adipose tissue (mWAT) in healthy and obese rats. Grape seed proanthocyanidin extracts (GSPEs) were administered for 21 days at 5, 25 or 50 mg GSPE/kg body weight in healthy rats and 25 mg GSPE/kg body weight in rats with diet-induced obesity. In healthy animals, GSPE administration led to the overexpression of core clock genes in a positive dose-dependent manner. Moreover, the acetylated BMAL1 protein ratio increased with the same pattern in the liver and mWAT. With regards to clock-controlled genes, Per2 was also overexpressed, whereas Rev-erbα and RORα were repressed in a negative dose-dependent manner. Diet-induced obesity always resulted in the overexpression of some core clock and clock-related genes, although the particular gene affected was tissue specific. GSPE administration counteracted disturbances in the clock genes in the liver and gut but was less effective in normalizing the clock gene disruption in WAT. In conclusion, proanthocyanidins have the capacity to modulate peripheral molecular clocks in both healthy and obese states.

Acute administration of grape seed proanthocyanidin extract modulates energetic metabolism in skeletal muscle and BAT mitochondria.

Proanthocyanidin consumption might reduce the risk of developing several pathologies, such as inflammation, oxidative stress and cardiovascular diseases. The beneficial effects of proanthocyanidins are attributed to their antioxidant properties, although they also can modulate gene expression at the transcriptional level. Little is known about the effect of proanthocyanidins on mitochondrial function and energy metabolism. In this context, the objective of this study was to determine the effect of an acute administration of grape seed proanthocyanidin extract (GSPE) on mitochondrial function and energy metabolism. To examine this effect, male Wistar rats fasted for fourteen hours, and then they were orally administered lard oil containing GSPE or were administered lard oil only. Liver, muscle and brown adipose tissue (BAT) were used to study enzymatic activity and gene expression of proteins related to energetic metabolism. Moreover, the gastrocnemius muscle and BAT mitochondria were used to perform high-resolution respirometry. The results showed that, after 5 h, the GSPE administration significantly lowers plasma triglycerides, free fatty acids, glycerol and urea concentrations. In skeletal muscle, GSPE lowers FATP1 mRNA levels and increases mitochondrial oxygen consumption, using pyruvate as the substrate, suggesting a promotion of glycosidic metabolism. Furthermore, GSPE increased the genetic expression of key genes in energy metabolism such as peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PGC1α), and modulated the enzyme activity of proteins, which are involved in the citric acid cycle and electron transport chain (ETC) in BAT. In conclusion, GSPE affects mainly the skeletal muscle and BAT mitochondria, increasing their oxidative capacity rapidly after acute supplementation.

Grape seed proanthocyanidins correct dyslipidemia associated with a high-fat diet in rats and repress genes controlling lipogenesis and VLDL assembling in liver.

OBJECTIVE: To determine whether proanthocyanidins can protect against dyslipidemia induced by a high-fat diet (HFD) and to address the mechanisms that underlie this hypolipidemic effect. DESIGN AND MEASUREMENTS: Female Wistar rats were fed on a HFD for 13 weeks. They were divided into two groups, one of which was treated with a grape seed proanthocyanidin extract (25 mg kg(-1) of body weight) for 10 days. Plasma and liver lipids were measured by colorimetric and gravimetric analysis. Liver, muscle and adipose tissue were used to study the expression of genes involved in the synthesis and oxidation of fatty acids and lipoprotein homeostasis by real-time RT-PCR. RESULTS: The administration of proanthocyanidins normalized plasma triglyceride and LDL-cholesterol (both parameters significantly increased with the HFD) but tended to decrease hypercholesterolemia and fatty liver. Gene expression analyses revealed that proanthocyanidins repressed both the expression of hepatic key regulators of lipogenesis and very low density lipoprotein (VLDL) assembling such as SREBP1, MTP and DGAT2, all of which were overexpressed by the HFD. CONCLUSION: These findings indicate that natural proanthocyanidins improve dyslipidemia associated with HFDs, mainly by repressing lipogenesis and VLDL assembly in the liver, and support the idea that they are powerful agents for preventing and treating lipid altered metabolic states.

Metabolic fate of glucose on 3T3-L1 adipocytes treated with grape seed-derived procyanidin extract (GSPE). Comparison with the effects of insulin.

In this paper we investigate the effects of a grape seed procyanidin extract (GSPE) on the metabolic fate of glucose in adipocytes. Differentiated 3T3-L1 cells were treated with 140 mg/L GSPE or 100 nM insulin for a short period (1 h, acute treatment) or for a long period (15 h, chronic treatment). 2-Deoxy-[1-(3)H]glucose uptake and [1-(14)C]glucose incorporation into cells, glycogen, and lipid were measured. We found that GSPE mimicked the anabolic effects of insulin but there were several important differences. GSPE stimulated glycogen synthesis less than insulin. After chronic exposure, GSPE induced a higher incorporation of glucose into lipid, mainly due to the increase in glucose directed to glycerol synthesis. Our main conclusions, therefore, are that GSPE has insulinomimetic properties and activates glycogen and lipid synthesis. However, the differences between the effects of GSPE and the effects of insulin indicate that GSPE uses mechanisms complementary to those of insulin signaling pathways to bring about these effects.

Grape-seed derived procyanidins interfere with adipogenesis of 3T3-L1 cells at the onset of differentiation.

OBJECTIVE: Our group's previous results on the effects of a grape seed procyanidin extract (GSPE) on adipose metabolism showed that peroxisome proliferator-activated receptor-gamma (PPARgamma) plays a central role in the lipolytic effects of GSPE on adipocytes. Since PPARgamma2 is a main regulator of the differentiation process of adipocytes, we investigated whether GSPE affects the adipogenesis of 3T3-L1 cells. DESIGN: We performed a time point screening by treating 3T3-L1 cells with GSPE during the differentiation process for 24 h. MEASUREMENTS: Differentiation markers and differential gene expression due to GSPE treatment (using the microarray technique). RESULTS: Twenty four hour-GSPE treatment at the onset of differentiation reduces adipose-specific markers and maintains the expression of preadipocyte marker preadipocyte factor-1 (Pref-1) significantly elevated. These effects were not found in other time points. Microarray analysis of gene expression after GSPE treatment at the early stage of differentiation showed a modified gene expression profile in which cell cycle and growth-related genes were downregulated by GSPE. CONCLUSION: These results suggest that GSPE affects adipogenesis, mainly at the induction of differentiation, and that procyanidins may have a new role in which they impede the formation of adipose cells.

Grape seed-derived procyanidins have an antihyperglycemic effect in streptozotocin-induced diabetic rats and insulinomimetic activity in insulin-sensitive cell lines.

Flavonoids are functional constituents of many fruits and vegetables. Some flavonoids have antidiabetic properties because they improve altered glucose and oxidative metabolisms of diabetic states. Procyanidins are flavonoids with an oligomeric structure, and it has been shown that they can improve the pathological oxidative state of a diabetic situation. To evaluate their effects on glucose metabolism, we administered an extract of grape seed procyanidins (PE) orally to streptozotocin-induced diabetic rats. This had an antihyperglycemic effect, which was significantly increased if PE administration was accompanied by a low insulin dose. The antihyperglycemic effect of PE may be partially due to the insulinomimetic activity of procyanidins on insulin-sensitive cell lines. PE stimulated glucose uptake in L6E9 myotubes and 3T3-L1 adipocytes in a dose-dependent manner. Like insulin action, the effect of PE on glucose uptake was sensitive to wortmannin, an inhibitor of phosphoinositol 3-kinase and to SB203580, an inhibitor of p38 MAPK. PE action also stimulated glucose transporter-4 translocation to the plasma membrane. In summary, procyanidins have insulin-like effects in insulin-sensitive cells that could help to explain their antihyperglycemic effect in vivo. These effects must be added to their antioxidant activity to explain why they can improve diabetic situations.

Nonalcoholic components in wine reduce low density lipoprotein cholesterol in normocholesterolemic rats.

Using an experimental model that enables the effects of alcohol to be distinguished from the effects of the nonalcoholic components present in wine, we determined whether wine has effects other than those of alcohol on the metabolism of cholesterol. Male rats were fed a standard diet and had free access to water and either wine or an equivalent alcohol solution for 45 d or 6 mon. Alcohol intake was similar in the two groups of animals. Consumption of the alcohol solution or wine did not influence plasma cholesterol or high density lipoprotein-cholesterol. At 45 d, the consumption both of wine and of alcohol solution reduced low density lipoprotein (LDL)-cholesterol and very low density lipoprotein cholesterol. At 6 mon, only the rats that consumed wine had reduced LDL-cholesterol. After 45 d of consuming alcohol solution, total cholesterol in the aorta was significantly increased mainly as a result of the rise in free cholesterol. In the aorta, the effect of wine consumption was similar to the effect of alcohol solution consumption, although it was less intense. The only clear effect that could be ascribed to the nonalcoholic components in wine was that the LDL-cholesterol was reduced in the long term, although aortic cholesterol was not.

Changes in lipolysis and hormone-sensitive lipase expression caused by procyanidins in 3T3-L1 adipocytes.

OBJECTIVE: To find out whether lipid stores are influenced by phenolic compounds in wine. DESIGN: Differentiated 3T3-L1 cells were treated with catechin, epicatechin or procyanidin extracts with different degrees of polymerization at 150 microM for different periods of time (0.5-24 h). SUBJECTS: Cell line 3T3-L1. MEASUREMENTS: Cellular viability, glycerol-3-phosphate dehydrogenase activity, glycerol release in the medium, HSL mRNA levels, triacylglycerols and protein. RESULTS: Catechin, epicatechin and procyanidin extracts were not toxic for the 3T3-L1 cells in the conditions assayed. Glycerol-3-phosphate dehydrogenase activity was markedly decreased by 150 microM procyanidin extracts. The release of glycerol into the medium was increased in 150 microM procyanidin extract-treated cells and reached a plateau after 15 h exposure. Procyanidins caused a time-dependent reduction in the HSL mRNA levels. CONCLUSIONS: These results suggest that procyanidins from grape and wine affect lipid metabolism whilst their monomers (catechin and epicatechin) do not. This effect is more pronounced when the degree of polymerization is higher. Procyanidin extracts cause a time-dependent reduction in the HSL mRNA levels, inhibit triacylglycerol synthesis and also favour triacylglycerol hydrolysis until the HSL mRNA had reached very low levels.

Moderate red wine consumption protects the rat against oxidation in vivo.

The effect of the moderate consumption of red wine on the antioxidant system in rat liver, kidney and plasma has been evaluated. Wistar rats were treated in separate groups as follows: control; red wine for 45 days or 6 months; and 13.5% ethanol for 45 days or 6 months. The consumption of alcoholic beverages was free because the rat could always choose between the alcoholic beverage and the water. In liver, red wine ingestion resulted in higher hepatic superoxide dismutase and glutathione peroxidase activities after 45 days of treatment. The data indicate that wine and ethanol ingestion resulted in lower hepatic malondialdehyde and enhanced hepatic catalase activity in both of the periods studied. In kidney, the reduced glutathione/oxidized glutathione ratio was higher after 45 days of wine consumption, and the malondialdehyde was lower after 6 months of wine consumption. In plasma, malondialdehyde was lower after 6 months of both treatments, but plasmatic vitamin E was higher after red wine consumption while it was lower after ethanol consumption for this period of time. The present study shows that the moderate and prolonged consumption of red wine is consistent with higher protection against oxidation in vivo.

Model for voluntary wine and alcohol consumption in rats.

It has been suggested that moderate consumption of ethanol and wine has a protective effect on human health. Animal models used to date for alcohol consumption can not mimic real situations in humans because the consumption is forced and/or excessive. The present study proposes to determine the effects of a voluntary and ad lib consumption model more similar to that of human behavior. Male Wistar rats had free access to either standard diet and water or the same diet plus red wine, sweet wine, or a solution equivalent to red wine (13.5% ethanol) or to sweet wine (20% ethanol + 130 g/L sucrose) for 30 days or 6 months. Daily wine consumption was 15.8 +/- 0.9 and 2.0 +/- 0.2 ml/day for sweet and red wines, respectively. The consumption of each of the alcoholic solutions was similar to that of the wine they were simulating. Drinking wine or ethanol did not affect food and water intakes or growth rate. Plasma metabolites were not substantially affected by consumption of wine or ethanol. Although moderate and high wine consumption did not change the activity of plasma marker enzymes of tissue damage, the consumption of the 2 alcoholic solutions caused a long-term increase in the activity of aspartate aminotransferase. It seems that wine consumption protects the organism from hepatic lesions induced by ethanol alone.

Plasma amino acids in hyperphagic pups subjected to a glucose gavage.

An oral gavage of either glucose or saline was given to pups fed either standard diet or cafeteria diet. The plasma amino acid concentrations were measured by a radiochemical method. In the standard diet group, plasma Asn+Asp, Thr, Pro, Cit, Trp and Phe levels were higher in rats receiving a glucose solution than in those given saline solution; taurine (on day 20) and Ser (on day 30) showed also higher plasma values. Plasma Arg and taurine levels in rats receiving glucose were lower than those in rats receiving saline when these pups were fed the cafeteria diet. Tyr (on day 20) and Gly and Pro (on day 30), showed decreased plasma values. The diet consumed during the days preceding a glucose gavage may have pronounced effects on several metabolites, particularly on nitrogen metabolism. The homeostasis of plasma amino acids was held highly constant in spite of the variety of diets supplied, indicating a remarkable homeostatic capacity on amino acidemia against dietetic manipulation.

Glutamine force-feeding effect on plasma amino-acid concentrations in growing rats fed a cafeteria diet.

The effect of a glutamine force-feeding on plasma amino-acid levels in rats fed either a reference diet or a cafeteria diet was studied during weaning. The increases in plasma amino-acid levels shown by rats eating the cafeteria diet were related to the force-feeding and/or the age studied. The glutamine solution decreased the levels of proline, ornithine and tyrosine in the plasma of rats eating the cafeteria diet. In rats fed the reference diet, glutamine solution increased the plasma concentrations of threonine and cysteine. A major effect of diet over force-feeding was shown.

The plasma amino acid response to cafeteria feeding and essential-amino acid gavage in weaning rats.

The plasma amino acid concentrations of cafeteria-fed and standard-fed rats gavaged either an essential amino acid mixture or saline solution have been studied from 14 to 30 days after birth. The consumption of a cafeteria diet caused higher levels in many amino acids. The amino acid-gavaged cafeteria-fed rats showed the highest cysteine levels. The amino acid gavage produced lower concentrations of alanine, glutamate+glutamine, hydroxyproline, proline and ornithine, in both cafeteria-fed and standard-fed animals. The results show that the supply of amino acids has a positive effect on nitrogen retention and amino acid availability in cafeteria-fed pups.

Postnatal development of plasma amino acids in hyperphagic rats.

The effect of feeding a highly palatable high-energy cafeteria diet on individual amino acid levels in plasma during postnatal development of the rat has been evaluated and compared to chow-fed controls. The cafeteria diet selected by the rats was hypercaloric and hyperlipidic, with practically the same amount of carbohydrate as the control diet, and slightly hyperproteic. In response to cafeteria feeding, significant decreases were observed in plasma serine and cysteine along the period studied. Significant changes with age during the growth period were shown by cafeteria-fed animals, which were not observed in control rats. Citrulline levels were lower on days 10 and 14 in cafeteria pups than in chow pups. Methionine was highest on day 30. Threonine was also higher at days 20 and 30, as was valine but with a nadir at day 10. Lysine showed maximal values on days 14 and 30.