Effects of flavonoids on intestinal inflammation, barrier integrity and changes in gut microbiota during diet-induced obesity.

Diet-induced obesity is associated with low-grade inflammation, which, in most cases, leads to the development of metabolic disorders, primarily insulin resistance and type 2 diabetes. Although prior studies have implicated the adipose tissue as being primarily responsible for obesity-associated inflammation, the latest discoveries have correlated impairments in intestinal immune homeostasis and the mucosal barrier with increased activation of the inflammatory pathways and the development of insulin resistance. Therefore, it is essential to define the mechanisms underlying the obesity-associated gut alterations to develop therapies to prevent and treat obesity and its associated diseases. Flavonoids appear to be promising candidates among the natural preventive treatments that have been identified to date. They have been shown to protect against several diseases, including CVD and various cancers. Furthermore, they have clear anti-inflammatory properties, which have primarily been evaluated in non-intestinal models. At present, a growing body of evidence suggests that flavonoids could exert a protective role against obesity-associated pathologies by modulating inflammatory-related cellular events in the intestine and/or the composition of the microbiota populations. The present paper will review the literature to date that has described the protective effects of flavonoids on intestinal inflammation, barrier integrity and gut microbiota in studies conducted using in vivo and in vitro models.

Nucleic acid lateral flow dipstick assay for the duplex detection of Gambierdiscus australes and Gambierdiscus excentricus.

The proliferation of harmful microalgae endangers aquatic ecosystems and can have serious economic implications on a global level. Harmful microalgae and their associated toxins also pose a threat to human health since they can cause seafood-borne diseases such as ciguatera. Implementation of DNA-based molecular methods together with appropriate detection strategies in monitoring programs can support the efforts for effective prevention of potential outbreaks. A PCR-lateral flow assay (PCR-LFA) in dipstick format was developed in this work for the detection of two Gambierdiscus species, G. australes and G. excentricus, which are known to produce highly potent neurotoxins known as ciguatoxins and have been associated with ciguatera outbreaks. Duplex PCR amplification of genomic DNA from strains of these species utilizing species-specific ssDNA tailed primers and a common primer containing the binding sequence of scCro DNA binding protein resulted in the generation of hybrid ssDNA-dsDNA amplicons. These were captured on the dipsticks via hybridization with complementary probes and detected with a scCro/carbon nanoparticle (scCro/CNPs) conjugate. The two different test zones on the dipsticks allowed the discrimination of the two species and the assay exhibited high sensitivity, 6.3 pg/µL of genomic DNA from both G. australes and G. excentricus. The specificity of the approach was also demonstrated using genomic DNA from non-target Gambierdiscus species and other microalgae genera which did not produce any signals. The possibility to use cells directly for amplification instead of purified genomic DNA suggested the compatibility of the approach with field sample testing. Future work is required to further explore the potential use of the strategy for on-site analysis and its applicability to other toxic species.

Glucagon Shows Higher Sensitivity than Insulin to Grapeseed Proanthocyanidin Extract (GSPE) Treatment in Cafeteria-Fed Rats.

The endocrine pancreas plays a key role in metabolism. Procyanidins (GSPE) targets ß-cells and glucagon-like peptide-1 (GLP-1)-producing cells; however, there is no information on the effects of GSPE on glucagon. We performed GSPE preventive treatments administered to Wistar rats before or at the same time as they were fed a cafeteria diet during 12 or 17 weeks. We then measured the pancreatic function and GLP-1 production. We found that glucagonemia remains modified by GSPE pre-treatment several weeks after the treatment has finished. The animals showed a higher GLP-1 response to glucose stimulation, together with a trend towards a higher GLP-1 receptor expression in the pancreas. When the GSPE treatment was administered every second week, the endocrine pancreas behaved differently. We show here that glucagon is a more sensitive parameter than insulin to GSPE treatments, with a secretion that is highly linked to GLP-1 ileal functionality and dependent on the type of treatment.

Grape Seed Proanthocyanidins Target the Enteroendocrine System in Cafeteria-Diet-Fed Rats.

SCOPE: The effects on the enteroendocrine system of three different grape seed proanthocyanidin extract (GSPE) treatments are analyzed in rats on a cafeteria diet for 17 weeks. METHODS AND RESULTS: GSPE is administered in a corrective manner (15 last days of the cafeteria diet) at two doses, 100 and 500 mg GSPE per kg bw. A third, longer treatment in which GSPE (500 mg kg-1 bw) is administered daily every other week during the 17 weeks of the cafeteria diet is also tested. Most GSPE treatments lead to ghrelin accumulation in the stomach, limited CCK secretion in the duodenum, and increased GLP-1 and PYY mRNA in colon. GSPE also increases cecal hypertrophy and reduces butyrate content. When the treatment is administered daily every other week during 17 weeks, there is also an increase in colon size. These effects are accompanied by a reduced food intake at the end of the experiment when GSPE is administered at 500 mg GSPE kg-1 during the last 15 days, but not on the other treatments, despite an observed reduction in body weight in the longer treatment. CONCLUSION: GSPE modulates the enteroendocrine system in models in which it also reduces food intake or body weight.

Grape-Seed Proanthocyanidins are Able to Reverse Intestinal Dysfunction and Metabolic Endotoxemia Induced by a Cafeteria Diet in Wistar Rats.

We evaluated the effectiveness of pharmacological doses of grape-seed proanthocyanidin extract (GSPE) in reversing intestinal barrier alterations and local inflammation in female Wistar rats fed a long-term obesogenic diet. Animals were fed a 17-week cafeteria diet (CAF diet), supplemented with daily GSPE doses (100 or 500 mg kg-1 body weight) during the final two weeks. CAF diet enhanced the intestinal permeation of an orally administered marker (ovalbumin, OVA) and increased the plasma levels of tumor necrosis factor-α (TNF-α) and lipopolysaccharides (LPS) in 2-3-fold. Ex vivo Ussing chamber assays showed a 55-70% reduction in transepithelial electrical resistance (TEER) and increased the TNF-α secretions in both small and large intestinal sections with a 25-fold increment in the ileum. Ileal tissues also presented a 4-fold increase of myeloperoxidase (MPO) activity. Both GSPE-treatments were able to restitute TEER values in the ileum and colon and to reduce plasma LPS to basal levels without a dose-dependent effect. However, effects on the OVA permeation and TNF-α secretion were dose and section-specific. GSPE also reduced ileal MPO activity and upregulated claudin 1 gene expression. This study provides evidence of the efficacy of GSPE-supplementation ameliorating diet-induced intestinal dysfunction and metabolic endotoxemia when administered at the end of a long-term obesogenic diet.

Protective Effect of Proanthocyanidins in a Rat Model of Mild Intestinal Inflammation and Impaired Intestinal Permeability Induced by LPS.

SCOPE: Intestinal dysfunction consists of a defective barrier function, which allows the influx of luminal endotoxins, thus causing intestinal inflammation. Proanthocyanidins are natural bioactive compounds that could modulate intestinal dysfunction. This study analyzes the protective effects of proanthocyanidins in a rat model of intestinal dysfunction. METHODS AND RESULTS: To investigate the preventive effects of both high dietary (75 mg kg-1 body weight) and pharmacological (375 mg kg-1 body weight) oral doses of proanthocyanidins (GSPE), rat intestinal dysfunction is induced with LPS (i.p.). In vivo intestinal permeability (ovalbumin [OVA] assay) and systemic inflammation and endotoxemia (TNF-α and LPS plasma levels) are assessed. Intestinal inflammation and oxidative stress are determined using myeloperoxidase (MPO), cyclooxygenase-2 (COX-2) activities, and reactive oxygen species (ROS) levels, respectively. Ileal gene expression of permeability/inflammatory genes is analyzed. LPS administration induces intestinal permeability, inflammation, and oxidative stress. GSPE normalizes in vivo OVA levels. In the small intestine, the GSPE treatment decreases MPO and COX-2 activities; modulates the ileum inflammatory and permeability proteins gene expression; and in the large intestine, prevents increase of ROS levels. CONCLUSIONS: Proanthocyanidins, at nutritional and pharmacological doses, prevents endotoxin-induced-intestinal inflammation, permeability, and oxidative stress in rats differentially in each intestinal section. Proanthocyanidins are nutritional-therapeutic novel candidates for preventing intestinal dysfunction.

Proanthocyanidins Limit Adipose Accrual Induced by a Cafeteria Diet, Several Weeks after the End of the Treatment.

A dose of proanthocyanidins with satiating properties proved to be able to limit body weight increase several weeks after administration under exposure to a cafeteria diet. Here we describe some of the molecular targets and the duration of the effects. We treated rats with 500 mg grape seed proanthocyanidin extract (GSPE)/kg BW for ten days. Seven or seventeen weeks after the last GSPE dose, while animals were on a cafeteria diet, we used reverse transcriptase-polymerase chain reaction (RT-PCR) to measure the mRNA of the key energy metabolism enzymes from the liver, adipose depots and muscle. We found that a reduction in the expression of adipose Lpl might explain the lower amount of adipose tissue in rats seven weeks after the last GSPE dose. The liver showed increased expression of Cpt1a and Hmgs2 together with a reduction in Fasn and Dgat2. In addition, muscle showed a higher fatty oxidation (Oxct1 and Cpt1b mRNA). However, after seventeen weeks, there was a completely different gene expression pattern. At the conclusion of the study, seven weeks after the last GSPE administration there was a limitation in adipose accrual that might be mediated by an inhibition of the gene expression of the adipose tissue Lpl. Concomitantly there was an increase in fatty acid oxidation in liver and muscle.

Long-Lasting Effects of GSPE on Ileal GLP-1R Gene Expression Are Associated with a Hypomethylation of the GLP-1R Promoter in Female Wistar Rats.

Flavonoids have been shown to modulate GLP-1 in obesity. GLP-1 induces some of its effects through the intestinal GLP-1 receptor (GLP-1R), though no data exist on how flavonoids affect this receptor. Here, we examine how a dose of grape seed proanthocyanidin extract (GSPE) with anti-obesity activity affects intestinal GLP-1R and analyze whether epigenetics play a role in the long-lasting effects of GSPE. We found that 10-day GSPE administration prior to the cafeteria diet upregulated GLP-1R mRNA in the ileum 17 weeks after the GSPE treatment. This was associated with a hypomethylation of the GLP-1R promoter near the region where the SP1 transcription factor binds. In the colon, the cafeteria diet upregulated GLP-1R without showing any GSPE effect. In conclusion, we have identified long-lasting GSPE effects on GLP-1R gene expression in the ileum that are partly mediated by hypomethylation at the gene promoter and may affect the SP1 binding factor.

The co-administration of proanthocyanidins and an obesogenic diet prevents the increase in intestinal permeability and metabolic endotoxemia derived to the diet.

The consumption of Westernized diets leads to hyperphagia and obesity, as well as intestinal alterations. In the present study, we evaluated the effect of the administration of a grape seed proanthocyanidin extract (GSPE) at different time points on the modulation of intestinal barrier function (intestinal permeability and metabolic endotoxemia), in rats with high-fat/high-carbohydrate diet-induced obesity. Animals were fed a cafeteria diet (CAF) supplemented with a preventive (PRE-CAF) or simultaneously intermittent (SIT-CAF) GSPE treatment (500 mg/kg bw). Changes in the plasma levels of an orally administered marker of intestinal permeability (ovalbumin, OVA), lipopolysaccharide (LPS) and tumor necrosis factor-α (TNF-α) were analyzed after animals were fed the obesogenic diet for 8, 12 and 17 weeks. In addition, ex vivo variations in transepithelial electrical resistance (TEER), the expression of tight junction (TJ) genes and the activity of myeloperoxidase (MPO) in the small and large intestines were monitored at the end of the experiment. The CAF diet increased OVA, LPS, MPO and TNF-α levels, accompanied by decreased TEER values in the small and large intestines. Interestingly, both GSPE treatments prevented these detrimental effects of the CAF diet, being the SIT-CAF group the most effective after 17 weeks of diet intervention. For the first time, this study provides evidence of the ameliorative effect of a proanthocyanidin extract, administered before or together with an obesogenic diet, on barrier dysfunction, as measured by intestinal permeability and metabolic endotoxemia.

Effects of an Intermittent Grape-Seed Proanthocyanidin (GSPE) Treatment on a Cafeteria Diet Obesogenic Challenge in Rats.

Obesity is highly associated with the pathologies included in the concept of the Metabolic Syndrome. Grape-seed proanthocyanins (GSPE) have showed very positive effects against all these metabolic disruptions; however, there is, as yet, no consensus about their effectiveness against an obesogenic challenge, such as a cafeteria diet. We determined the effectiveness of a dose of 500 mg GSPE/kg b.w. (body weight) against the obesogenic effects of a 17-week cafeteria diet, administered as a sub-chronic treatment, 10-15 days before, intermittently and at the end of the diet, in Wistar rats. Body weight, adiposity, indirect calorimetry and plasma parameters were analyzed. GSPE pre-treatment showed a long-lasting effect on body weight and adiposity that was maintained for seven weeks after the last dose. A corrective treatment was administered for the last two weeks of the cafeteria diet intervention; however, it did not effectively correct any of the parameters assessed. The most effective treatment was an intermittent GSPE dosage, administered every second week during the cafeteria diet. This limited body weight gain, adiposity and most lipotoxic effects. Our results support the administration of this GSPE dose, keeping an intermittent interval between dosages longer than every second week, to improve obesogenic disruptions produced by a cafeteria diet.

Novel ex Vivo Experimental Setup to Assay the Vectorial Transepithelial Enteroendocrine Secretions of Different Intestinal Segments.

The enteroendocrine system coordinates gastrointestinal (GI) tract functionality and the whole organism. However, the scarcity of enteroendocrine cells and their scattered distribution make them difficult to study. Here, we glued segments of the GI wall of pigs to a silicon tube, keeping the apical and the basolateral sides separate. The fact that there was less than 1% of 70-kDa fluorescein isothiocyanate (FITC)-dextran on the basolateral side proved that the gluing was efficient. Since the lactate dehydrogenase leakage at basolateral side was lower than 0.1% (1.40 ± 0.17 nKatals) it proved that the tissue was viable. The intestinal barrier function was maintained as it is in segments mounted in Ussing chambers (the amount of Lucifer Yellow crossing it, was similar between them; respectively, % LY, 0.48 ± 0.13; 0.52 ± 0.09; p > 0.05). Finally, apical treatments with two different extract produced differential basolateral enterohormone secretions (basolateral PYY secretion vs control; animal extract, 0.35 ± 0.16; plant extract, 2.5 ± 0.74; p < 0.05). In conclusion, we report an ex vivo system called "Ap-to-Bas" for assaying vectorial transepithelial processes that makes it possible to work with several samples at the same time. It is an optimal device for enterohormone studies in the intestine.

Chronic supplementation with dietary proanthocyanidins protects from diet-induced intestinal alterations in obese rats.

SCOPE: Increased attention has been paid to the link between altered intestinal function and elevated incidence of metabolic disorders, such as in obesity. This study investigated in obese rats the role of grape seed proanthocyanidin extract (GSPE) chronic treatment, taken in a low, moderate, or high dose, on obesity-associated intestinal alterations in response to a cafeteria diet (CAF). METHODS AND RESULTS: To evaluate the degree of intestinal inflammation, reactive oxygen species (ROS) production and myeloperoxidase (MPO) activity were measured as well as the expression of inflammatory-related genes. The barrier integrity was assessed by quantifying the gene expression of tight-junction components and measuring the plasma LPS. GSPE decreased the ROS levels and MPO activity, without substantial differences among the doses. The supplementation with moderate and high GSPE doses significantly decreased iNOS expression compared to the CAF group, and the same pattern was observed in the low-dose animals with respect to IL-1ß expression. Moreover, the results show that GSPE significantly increases zonulin-1 expression with respect to the CAF animals. CONCLUSION: This study provides evidence for the ameliorative effect of a proanthocyanidin extract on high-fat/high-carbohydrate diet-induced intestinal alterations, specifically reducing intestinal inflammation and oxidative stress and suggesting a protection against a barrier defect.