Gaiping Wen

Supplementation of a grape seed and grape marc meal extract decreases activities of the oxidative stress-responsive transcription factors NF-κB and Nrf2 in the duodenal mucosa of pigs

BackgroundIn pigs, enteric infections and the development of gut disorders such as diarrhoea are commonly observed, particularly after weaning. The present study investigated the hypothesis that feeding a grape seed and grape marc extract (GSGME) as a dietary supplement has the potential to suppress the inflammatory process in the small intestine of pigs by modulating the activities of NF-κB and Nrf2 due to its high content of flavonoids.MethodsTwenty-four crossbred, 6 weeks old pigs were randomly assigned to 2 groups of 12 animals each and fed nutritionally adequate diets without or with 1% GSGME for 4 weeks.ResultsPigs administered GSGME had a lower transactivation of NF-κB and Nrf2 and a lower expression of various target genes of these transcription factors in the duodenal mucosa than control pigs (P < 0.05). Concentrations of α-tocopherol and thiobarbituric acid reactive substances (TBARS) in liver and plasma and total antioxidant capacity of plasma and relative mRNA abundances of NF-κB and Nrf2 target genes in the liver did not differ between the two groups. However, the ratio of villus height:crypt depth and the gain:feed ratio was higher in the pigs fed GSGME than in control pigs (P < 0.05).ConclusionsThis study shows that dietary supplementation of a polyphenol rich GSGME suppresses the activity of NF-κB in the duodenal mucosa of pigs and thus might provide a useful dietary strategy to inhibit inflammation in the gut frequently occurring in pigs. Feeding GSGME did not influence vitamin E status and the antioxidant system of the pigs but improved the gain:feed ratio. In overall, the study suggests that polyphenol-rich plant extracts such GSGME could be useful feed supplements in pig nutrition, in order to maintain animal health and improve performance.

Inhibition of the pro‐inflammatory NF‐κB pathway by a grape seed and grape marc meal extract in intestinal epithelial cells

In pigs and other monogastric animal, the weaning phase is commonly accompanied by an increased susceptibility to gut disorders such as diarrhoea owing to the induction of an inflammatory process in the intestine during weaning. Given the unfavourable effects of intestinal inflammation on feed consumption, digestive capacity of the intestine and growth of animals, controlling intestinal inflammation is a reasonable approach for the maintenance of performance characteristics of livestock animals. Therefore, this study aimed to study the anti-inflammatory potential of a commercial polyphenol-rich grape seed (GS) and grape marc (GM) meal-based feed additive in a well-established in vitro intestinal epithelium model (polarized Caco-2 cells). The anti-inflammatory potential was evaluated by studying the effect of an ethanolic extract obtained from the GS and GM meal-based feed additive (GSGME) on the pro-inflammatory transcription factor NF-κB, which is considered to play a key role in the induction of weaning-associated intestinal inflammation. The highest non-cytotoxic concentrations of the ethanolic GSGME dose dependently reduced TNFα-induced NF-κB transactivation and decreased TNFα-induced mRNA levels of the NF-κB target genes IL-1β, IL-8, MCP-1 and CXCL1 in Caco-2 intestinal cells (p < 0.05). No effect of the ethanolic GSGME was observed on the cytoprotective Nrf2 pathway in Caco-2 cells as evidenced by an unaltered Nrf2 transactivation and unchanged mRNA levels of Nrf2 target genes, such as GPX-2, NQO1, CYP1A1 and UGT1A1. In conclusion, this study shows that an ethanolic GSGME exerts anti-inflammatory effects in intestinal cells under in vitro conditions. Thus, polyphenol-rich GSGM meal-based feed additives may be useful for the inhibition or prevention of inflammatory processes in the intestine of livestock animals, in particular during states with inappropriate NF-κB activation in the intestinal tissue, such as the weaning phase. Future studies are warranted to prove the in vivo anti-inflammatory potential of GSGM meal-based feed additives.

Regular endurance exercise improves the diminished hepatic carnitine status in mice fed a high-fat diet

SCOPE Metabolic stress induced by chronic high-fat (HF) diet feeding or genetically induced diabetes impairs carnitine status. Herein, we tested the hypothesis that regular endurance exercise (EE) improves the HF diet-induced impairment of carnitine status through stimulating the expression of hepatic genes involved in carnitine synthesis and uptake. METHODS AND RESULTS Eighteen male C57BL/6 mice were assigned to three groups: group S received a standard diet, group HF received a HF diet, and group HF+EE received an HF diet and was regularly exercised on a treadmill. After 10 wk, mice of the HF and the HF+EE groups were highly obese and insulin resistant compared with mice of the S group (p<0.05), but mice of the HF+EE group were less insulin resistant than those of the HF group (p<0.05). The HF group had lower carnitine concentrations and mRNA and protein levels of genes involved in carnitine synthesis and uptake in the liver than the S group (p<0.05), whereas these parameters did not differ between the S group and the HF+EE group. CONCLUSION These findings indicate that regular EE reverses an HF diet-induced impairment of hepatic carnitine content by stimulating hepatic carnitine synthesis and uptake.

13-hydroxy linoleic acid increases expression of the cholesterol transporters ABCA1, ABCG1 and SR-BI and stimulates apoA-I-dependent cholesterol efflux in RAW264.7 macrophages

BackgroundSynthetic activators of peroxisome proliferator-activated receptors (PPARs) stimulate cholesterol removal from macrophages through PPAR-dependent up-regulation of liver × receptor α (LXRα) and subsequent induction of cholesterol exporters such as ATP-binding cassette transporter A1 (ABCA1) and scavenger receptor class B type 1 (SR-BI). The present study aimed to test the hypothesis that the hydroxylated derivative of linoleic acid (LA), 13-HODE, which is a natural PPAR agonist, has similar effects in RAW264.7 macrophages.MethodsRAW264.7 macrophages were treated without (control) or with LA or 13-HODE in the presence and absence of PPARα or PPARγ antagonists and determined protein levels of LXRα, ABCA1, ABCG1, SR-BI, PPARα and PPARγ and apolipoprotein A-I mediated lipid efflux.ResultsTreatment of RAW264.7 cells with 13-HODE increased PPAR-transactivation activity and protein concentrations of LXRα, ABCA1, ABCG1 and SR-BI when compared to control treatment (P < 0.05). In addition, 13-HODE enhanced cholesterol concentration in the medium but decreased cellular cholesterol concentration during incubation of cells with the extracellular lipid acceptor apolipoprotein A-I (P < 0.05). Pre-treatment of cells with a selective PPARα or PPARγ antagonist completely abolished the effects of 13-HODE on cholesterol efflux and protein levels of genes investigated. In contrast to 13-HODE, LA had no effect on either of these parameters compared to control cells.Conclusion13-HODE induces cholesterol efflux from macrophages via the PPAR-LXRα-ABCA1/SR-BI-pathway.

The mouse gene encoding the carnitine biosynthetic enzyme 4-N-trimethylaminobutyraldehyde dehydrogenase is regulated by peroxisome proliferator-activated receptor α.

Genes involved in carnitine uptake and synthesis, such as organic cation transporter-2 (OCTN2) and γ-butyrobetaine dioxygenase (BBD), have been shown to be regulated by peroxisome proliferator-activated receptor (PPAR)α directly. Whether other genes encoding enzymes involved in the carnitine synthesis pathway, such as 4-N-trimethylaminobutyraldehyde dehydrogenase (TMABA-DH) and trimethyllysine dioxygenase (TMLD), are also direct PPARα target genes is less clear. In silico-analysis of the mouse TMLD promoter and first intron and the TMABA-DH promoter revealed several putative peroxisome proliferator response elements (PPRE) with high similarity to the consensus PPRE. Luciferase reporter gene assays using either a 2kb TMLD promoter or a 4kb TMLD first intron reporter constructs revealed no functional PPRE. In contrast, reporter gene assays using wild-type and mutated 5´-truncation TMABA-DH promoter reporter constructs showed that one PPRE located at position -132 in the proximal promoter is probably functional. Using gel shift assays we observed in vitro-binding of PPARα to this PPRE. Moreover, using chromatin immunoprecipitation assays we found that PPARα also binds in vivo to a nucleotide sequence spanning the PPRE at -132, which confirms that this PPRE is functional. In conclusion, the present study shows that the mouse TMABA-DH gene is a direct PPARα target gene. Together with the recent identification of the mouse BBD and the mouse OCTN2 genes as PPARα target genes this finding confirm that PPARα plays a key role in the regulation of carnitine homeostasis by controlling genes involved in carnitine synthesis and carnitine uptake.

Treatment with pharmacological PPARα agonists stimulates the ubiquitin proteasome pathway and myofibrillar protein breakdown in skeletal muscle of rodents.

BACKGROUND Treatment of hyperlipidemic patients with fibrates, agonists of peroxisome proliferator-activated receptor α (PPARα), provokes muscle atrophy as a side effect. The molecular mechanism underlying this phenomenon is still unknown. We tested the hypothesis that activation of PPARα leads to an up-regulation of the ubiquitin proteasome system (UPS) which plays a major role in protein degradation in muscle. METHODS Rats, wild-type and PPARα-deficient mice (PPARα(-/-)) were treated with synthetic PPARα agonists (clofibrate, WY-14,643) to study their effect on the UPS and myofibrillar protein breakdown in muscle. RESULTS In rats and wild-type mice but not PPARα(-/-) mice, clofibrate or WY-14,643 caused increases in mRNA and protein levels of the ubiquitin ligases atrogin-1 and MuRF1 in muscle. Wild-type mice treated with WY-14,643 had a greater 3-methylhistidine release from incubated muscle and lesser muscle weights. In addition, wild-type mice but not PPARα(-/-) mice treated with WY-14,643 had higher amounts of ubiquitin-protein conjugates, a decreased activity of PI3K/Akt1 signalling, and an increased activity of FoxO1 transcription factor in muscle. Reporter gene and gel shift experiments revealed that the atrogin-1 and MuRF1 promoter do not contain functional PPARα DNA-binding sites. CONCLUSIONS These findings indicate that fibrates stimulate ubiquitination of proteins in skeletal muscle which in turn stimulates protein degradation. Up-regulation of ubiquitin ligases is probably not mediated by PPARα-dependent gene transcription but by PPARα-dependent inhibition of the PI3K/Akt1 signalling pathway leading to activation of FoxO1. GENERAL SIGNIFICANCE PPARα plays a role in the regulation of the ubiquitin proteasome system.

Short communication: The pharmacological peroxisome proliferator-activated receptor α agonist WY-14,643 increases expression of novel organic cation transporter 2 and carnitine uptake in bovine kidney cells

Recent studies in rodents demonstrated that peroxisome proliferator-activated receptor α (PPARα), a central regulator of energy homeostasis, is an important transcriptional regulator of the gene encoding the carnitine transporter novel organic cation transporter 2 (OCTN2). Less is known with regard to the regulation of OCTN2 by PPARα and its role for carnitine transport in cattle, even though PPARα activation physiologically occurs in the liver of high-producing cows during early lactation. To explore the role of PPARα for OCTN2 expression and carnitine transport in cattle, we studied the effect of the PPARα activator WY-14,643 on the expression of OCTN2 in the presence and absence of PPARα antagonists and on OCTN2-mediated carnitine transport in the Madin-Darby bovine kidney (MDBK) cell line. The results show that WY-14,643 increases mRNA and protein levels of OCTN2, whereas co-treatment of MDBK cells with WY-14,643 and the PPARα antagonist GW6471 blocks the WY-14,643-induced increase in mRNA and protein levels of OCTN2 in bovine cells. In addition, treatment of MDBK cells with WY-14,643 stimulates specifically Na(+)-dependent carnitine uptake in MDBK cells, which is likely the consequence of the increased carnitine transport capacity of cells due to the elevated expression of OCTN2. In conclusion, our results indicate that OCTN2 expression and carnitine transport in cattle, as in rodents, are regulated by PPARα.

Transcriptional regulation of the human, porcine and bovine OCTN2 gene by PPARα via a conserved PPRE located in intron 1

BackgroundThe novel organic cation transporter 2 (OCTN2) is the physiologically most important carnitine transporter in tissues and is responsible for carnitine absorption in the intestine, carnitine reabsorption in the kidney and distribution of carnitine between tissues. Genetic studies clearly demonstrated that the mouse OCTN2 gene is directly regulated by peroxisome proliferator-activated receptor α (PPARα). Despite its well conserved role as an important regulator of lipid catabolism in general, the specific genes under control of PPARα within each lipid metabolic pathway were shown to differ between species and it is currently unknown whether the OCTN2 gene is also a PPARα target gene in pig, cattle, and human. In the present study we examined the hypothesis that the porcine, bovine, and human OCTN2 gene are also PPARα target genes.ResultsUsing positional cloning and reporter gene assays we identified a functional PPRE, each in the intron 1 of the porcine, bovine, and human OCTN2 gene. Gel shift assay confirmed binding of PPARα to this PPRE in the porcine, bovine, and the human OCTN2 gene.ConclusionsThe results of the present study show that the porcine, bovine, and human OCTN2 gene, like the mouse OCTN2 gene, is directly regulated by PPARα. This suggests that regulation of genes involved in carnitine uptake by PPARα is highly conserved across species.

The pro-inflammatory cytokine tumor necrosis factor α stimulates expression of the carnitine transporter OCTN2 (novel organic cation transporter 2) and carnitine uptake via nuclear factor-κB in Madin-Darby bovine kidney cells

Carnitine uptake into tissues is mediated mainly by the novel organic cation transporter 2 (OCTN2), whose expression is upregulated in the liver of early-lactating dairy cows. It has been shown recently that pro-inflammatory cytokines, including tumor necrosis factor α (TNFα), stimulate OCTN2 expression and carnitine uptake in intestinal cells and inflamed intestinal mucosa. Given that many early-lactating dairy cows show typical signs of hepatic and systemic inflammation, such as elevated concentrations of circulating TNFα and activation of the key regulator of inflammation, nuclear factor κB (NF-κB), in tissues, it is possible that upregulation of OCTN2 and increase of carnitine uptake by TNFα is mediated by NF-κB, a mechanism that might contribute to the upregulation of OCNT2 in the liver of early-lactating dairy cows. Thus, in the present study, we tested the hypothesis that TNFα stimulates OCTN2 gene expression and carnitine uptake via NF-κB in the bovine Madin-Darby bovine kidney (MDBK) cell line. Treatment with TNFα caused activation of NF-κB, increased the mRNA and protein concentration of OCTN2, and stimulated the uptake of carnitine in MDBK cells. In contrast, combined treatment of MDBK cells with TNFα and the NF-κB inhibitor BAY 11-7085 completely blocked the effect of TNFα on OCTN2 mRNA and protein concentration and uptake of carnitine. These findings suggest that the bovine OCTN2 gene and carnitine uptake are regulated by NF-κB. Future studies are required to show the in vivo relevance of this regulatory mechanism in cattle.

Carnitine transporter OCTN2 and carnitine uptake in bovine kidney cells is regulated by peroxisome proliferator-activated receptor β/δ

BackgroundPeroxisome proliferator-activated receptor α (PPARα), a central regulator of fatty acid catabolism, has recently been shown to be a transcriptional regulator of the gene encoding the carnitine transporter novel organic cation transporter 2 (OCTN2) in cattle. Whether PPARβ/δ, another PPAR subtype, which has partially overlapping functions as PPARα and is known to share a large set of common target genes with PPARα, also regulates OCTN2 and carnitine transport in cattle is currently unknown. To close this gap of knowledge, we studied the effect of the PPARβ/δ activator GW0742 on mRNA and protein levels of OCTN2 and carnitine uptake in the presence and absence of the PPARβ/δ antagonist GSK3787 in the bovine Madin-Darby bovine kidney (MDBK) cell line.FindingsTreatment of MDBK cells with GW0742 caused a strong increase in the mRNA level of the known bovine PPARβ/δ target gene CPT1A in MDBK cells indicating activation of PPARβ/δ. The mRNA and protein level of OCTN2 was clearly elevated in MDBK cells treated with GW0742, but the stimulatory effect of GW0742 on mRNA and protein level of OCTN2 was completely blocked by GSK3787. In addition, GW0742 increased Na+-dependent carnitine uptake, which is mediated by OCTN2, into MDBK cells, whereas treatment of cells with the PPARβ/δ antagonist completely abolished the stimulatory effect of GW0742 on carnitine uptake.ConclusionsThe present study shows for the first time that gene expression of the carnitine transporter OCTN2 and carnitine transport are regulated by PPARβ/δ in bovine cells. These novel findings extend the knowledge about the molecular regulation of the OCTN2 gene and carnitine transport in cattle and indicate that regulation of OCTN2 gene expression and carnitine transport is not restricted to the PPARα subtype.