Time-of-Day Circadian Modulation of Grape-Seed Procyanidin Extract (GSPE) in Hepatic Mitochondrial Dynamics in Cafeteria-Diet-Induced Obese Rats.

Major susceptibility to alterations in liver function (e.g., hepatic steatosis) in a prone environment due to circadian misalignments represents a common consequence of recent sociobiological behavior (i.e., food excess and sleep deprivation). Natural compounds and, more concisely, polyphenols have been shown as an interesting tool for fighting against metabolic syndrome and related consequences. Furthermore, mitochondria have been identified as an important target for mediation of the health effects of these compounds. Additionally, mitochondrial function and dynamics are strongly regulated in a circadian way. Thus, we wondered whether some of the beneficial effects of grape-seed procyanidin extract (GSPE) on metabolic syndrome could be mediated by a circadian modulation of mitochondrial homeostasis. For this purpose, rats were subjected to "standard", "cafeteria" and "cafeteria diet + GSPE" treatments (n = 4/group) for 9 weeks (the last 4 weeks, GSPE/vehicle) of treatment, administering the extract/vehicle at diurnal or nocturnal times (ZT0 or ZT12). For circadian assessment, one hour after turning the light on (ZT1), animals were sacrificed every 6 h (ZT1, ZT7, ZT13 and ZT19). Interestingly, GSPE was able to restore the rhythm on clock hepatic genes (Bmal1, Per2, Cry1, Rorα), as this correction was more evident in nocturnal treatment. Additionally, during nocturnal treatment, an increase in hepatic fusion genes and a decrease in fission genes were observed. Regarding mitochondrial complex activity, there was a strong effect of cafeteria diet at nearly all ZTs, and GSPE was able to restore activity at discrete ZTs, mainly in the diurnal treatment (ZT0). Furthermore, a differential behavior was observed in tricarboxylic acid (TCA) metabolites between GSPE diurnal and nocturnal administration times. Therefore, GSPE may serve as a nutritional preventive strategy in the recovery of hepatic-related metabolic disease by modulating mitochondrial dynamics, which is concomitant to the restoration of the hepatic circadian machinery.

Dietary proanthocyanidins modulate melatonin levels in plasma and the expression pattern of clock genes in the hypothalamus of rats.

SCOPE: Circadian rhythms allow organisms to anticipate and adapt to environmental changes, and food components can adjust internal rhythms. Proanthocyanidins improve cardiovascular risk factors that exhibit circadian oscillations. Therefore, the aim of the current study was to determine whether proanthocyanidins can modulate body rhythms. METHODS AND RESULTS: Male Wistar rats were orally gavaged with 250 mg grape seed proanthocyanidin extract (GSPE)/kg body weight at zeitgeber time (ZT) 0 (light on). Phenotypic biorhythm was evaluated by measuring the concentration of plasma melatonin and metabolites, using MNR-metabolomics, at several ZT. Remarkably, GSPE treatment maintained nocturnal melatonin levels at ZT3 and altered the oscillations of some metabolites in plasma. Quantification of expression of clock-core (Clock, Bmal1, Per2, Rorα, Rev-erbα) and clock-controlled (Nampt) genes in the hypothalamus by RT-PCR showed that this phenotypic alteration was concomitant with the modulation of the expression pattern of Bmal1 and Nampt. However, GSPE did not modulate the nocturnal expression of clock genes when administered at ZT12 (light off). CONCLUSION: PAs could have chronobiological properties, although their activity depends largely on the time of administration.

Grape seed proanthocyanidin extract improves the hepatic glutathione metabolism in obese Zucker rats.

SCOPE: Increased oxidative stress may play an important role in metabolic syndrome and related manifestations, including obesity, atherosclerosis, hypertension, and insulin resistance. Its relation to obesity is due to increased reactive oxygen species and/or decreased glutathione (GSH) antioxidant metabolism. Consequently, the activation of glutathione metabolism appears to be a central defense response to prevent oxidative stress. In this sense, dietary supplements with natural antioxidant molecules, including proanthocyanidins, may present a useful strategy of controlling and reducing complications of obesity, including hepatic steatosis. MATERIALS AND RESULTS: We assessed the grape seed proanthocyanidin extract (GSPE) effect on oxidative alterations related to genetically obese rats (Zucker rats) and, more specifically, to hepatic GSH metabolism. We demonstrate that the administration of GSPE reduced the oxidized glutathione accumulation increasing the total GSH/oxidized glutathione hepatic ratio and consequently decreasing the activation of antioxidant enzymes, including glutathione peroxidase, glutathione reductase, and glutathione S-transferase, and increasing the total antioxidant capacity of the cell. CONCLUSION: In Zucker rats, the obesity-induced oxidative stress related to liver glutathione alteration was mitigated by GSPE administration.

Grape seed procyanidin extract reduces the endotoxic effects induced by lipopolysaccharide in rats.

Acute inflammation is a response to injury, infection, tissue damage, or shock. Bacterial lipopolysaccharide (LPS) is an endotoxin implicated in triggering sepsis and septic shock, and LPS promotes the inflammatory response, resulting in the secretion of proinflammatory and anti-inflammatory cytokines such as the interleukins (IL-6, IL-1ß, and IL-10) and tumor necrosis factor-α by the immune cells. Furthermore, nitric oxide and reactive oxygen species levels increase rapidly, which is partially due to the activation of inducible nitric oxide synthase in several tissues in response to inflammatory stimuli. Previous studies have shown that procyanidins, polyphenols present in foods such as apples, grapes, cocoa, and berries, have several beneficial properties against inflammation and oxidative stress using several in vitro and in vivo models. In this study, the anti-inflammatory and antioxidant effects of two physiological doses and two pharmaceutical doses of grape seed procyanidin extract (GSPE) were analyzed using a rat model of septic shock by the intraperitoneal injection of LPS derived from Escherichia coli. The high nutritional (75mg/kg/day) and the high pharmacological doses (200mg/kg/day) of GSPE showed anti-inflammatory effects by decreasing the proinflammatory marker NOx in the plasma, red blood cells, spleen, and liver. Moreover, the high pharmacological dose also downregulated the genes Il-6 and iNos; and the high nutritional dose decreased the glutathione ratio (GSSG/total glutathione), further illustrating the antioxidant capability of GSPE. In conclusion, several doses of GSPE can alleviate acute inflammation triggered by LPS in rats at the systemic and local levels when administered for as few as 15 days before the injection of endotoxin.

Identification of PPARgamma partial agonists of natural origin (II): in silico prediction in natural extracts with known antidiabetic activity.

BACKGROUND: Natural extracts have played an important role in the prevention and treatment of diseases and are important sources for drug discovery. However, to be effectively used in these processes, natural extracts must be characterized through the identification of their active compounds and their modes of action. METHODOLOGY/PRINCIPAL FINDINGS: From an initial set of 29,779 natural products that are annotated with their natural source and using a previously developed virtual screening procedure (carefully validated experimentally), we have predicted as potential peroxisome proliferators-activated receptor gamma (PPARγ) partial agonists 12 molecules from 11 extracts known to have antidiabetic activity. Six of these molecules are similar to molecules with described antidiabetic activity but whose mechanism of action is unknown. Therefore, it is plausible that these 12 molecules could be the bioactive molecules responsible, at least in part, for the antidiabetic activity of the extracts containing them. In addition, we have also identified as potential PPARγ partial agonists 10 molecules from 16 plants with undescribed antidiabetic activity but that are related (i.e., they are from the same genus) to plants with known antidiabetic properties. None of the 22 molecules that we predict as PPARγ partial agonists show chemical similarity with a group of 211 known PPARγ partial agonists obtained from the literature. CONCLUSIONS/SIGNIFICANCE: Our results provide a new hypothesis about the active molecules of natural extracts with antidiabetic properties and their mode of action. We also suggest plants with undescribed antidiabetic activity that may contain PPARγ partial agonists. These plants represent a new source of potential antidiabetic extracts. Consequently, our work opens the door to the discovery of new antidiabetic extracts and molecules that can be of use, for instance, in the design of new antidiabetic drugs or functional foods focused towards the prevention/treatment of type 2 Diabetes Mellitus.

Identification of human IKK-2 inhibitors of natural origin (Part II): in Silico prediction of IKK-2 inhibitors in natural extracts with known anti-inflammatory activity.

Human inhibitor NF-κB kinase 2 (hIKK-2) is the primary component responsible for activating NF-κB in response to various inflammatory stimuli. Thus, synthetic ATP-competitive inhibitors for hIKK-2 have been developed as anti-inflammatory compounds. We recently reported a virtual screening protocol (doi:10.1371/journal.pone.0016903) that is able to identify hIKK-2 inhibitors that are not structurally related to any known molecule that inhibits hIKK-2 and that have never been reported to have anti-inflammatory activity. In this study, a stricter version of this protocol was applied to an in-house database of 29,779 natural products annotated with their natural source. The search identified 274 molecules (isolated from 453 different natural extracts) predicted to inhibit hIKK-2. An exhaustive bibliographic search revealed that anti-inflammatory activity has been previously described for: (a) 36 out of these 453 extracts; and (b) 17 out of 30 virtual screening hits present in these 36 extracts. Only one of the remaining 13 hit molecules in these extracts shows chemical similarity with known synthetic hIKK-2 inhibitors. Therefore, it is plausible that a significant portion of the remaining 12 hit molecules are lead-hopping candidates for the development of new hIKK-2 inhibitors.

Hypolipidemic effects of proanthocyanidins and their underlying biochemical and molecular mechanisms.

Proanthocyanidins are the most abundant polyphenols in human diets. Epidemiological studies strongly suggest that proanthocyanidins protect against cardiovascular diseases. Despite the antioxidant and anti-inflammatory properties of these flavonoids, one of the mechanisms by which proanthocyanidins exert their cardiovascular protection is improving lipid homeostasis. Animal studies demonstrate that proanthocyanidins reduce the plasma levels of atherogenic apolipoprotein B-triglyceride-rich lipoproteins and LDL-cholesterol but increase antiatherogenic HDL-cholesterol. The results in humans, however, are less clear. This review summarizes the results that have been published on plasma triglyceride, apolipoprotein B, HDL-cholesterol and LDL-cholesterol levels in humans and animal models in response to proanthocyanidin extracts and proanthocyanidin-rich foods. The physiological processes and biochemical pathways that are related to lipid homeostasis and affected by proanthocyanidin consumption are also discussed. Intestinal lipid absorption, chylomicron secretion by the intestine and VLDL secretion by the liver are the processes that are most repressed by proanthocyanidins, which, therefore, induce hypolipidemic effects.

Dietary procyanidins lower triglyceride levels signaling through the nuclear receptor small heterodimer partner.

Hypertriglyceridemia is an independent risk factor in the development of cardiovascular diseases, and we have previously reported that oral administration of a grape seed procyanidin extract (GSPE) drastically decreases plasma levels of triglycerides (TG) and apolipoprotein B (ApoB) in normolipidemic rats, with a concomitant induction in the hepatic expression of the nuclear receptor small heterodimer partner (NR0B2/SHP). Our objective in this study was to elucidate whether SHP is the mediator of the reduction of TG-rich ApoB-containing lipoproteins triggered by GSPE. We show that GSPE inhibited TG and ApoB secretion in human hepatocarcinoma HepG2 cells and had and hypotriglyceridemic effect in wild-type mouse. The TG-lowering action of GSPE was abolished in HepG2 cells transfected with a SHP-specific siRNA and in a SHP-null mouse. Moreover, in mouse liver, GSPE downregulated several lipogenic genes, including steroid response element binding protein 1c (SREBP-1c), and upregulated carnitine palmitoyltransferase-1A (CPT-1A) and apolipoprotein A5 (ApoA5), in a SHP-dependent manner. In HepG2 cells GSPE also inhibited ApoB secretion, but in a SHP-independent manner. In conclusion, SHP is a key mediator of the hypotriglyceridemic response triggered by GSPE. This novel signaling pathway of procyanidins through SHP may be relevant to explain the health effects ascribed to the regular consumption of dietary flavonoids.