Effects of Escherichia coli strain Nissle 1917 on exercise-induced disruption of gastrointestinal integrity.

PURPOSE: The aim of the current study was to investigate the effects of the probiotic Escherichia coli strain Nissle 1917 (EcN) on the exercise-induced disruption of gastrointestinal (GI) integrity and the associated release of damage and inflammatory markers. METHODS: After a pre-performance test, 19 untrained subjects (aged 18-35 years) passed two identical exhaustive treadmill exercise tests in an intensity corresponding to 60-80% VO2max in a test-retest design. The exercise tests were separated by a time period of 4 weeks. During this period, all subjects ingested 5 ml of an EcN suspension daily. Serum samples were taken before, immediately following and 3 h after both exercise tests. They were analyzed for indicators of GI integrity (zonulin; claudin-3; LPS), various damage and redox markers (I-FABP, GOT; GPT; TBARS) and inflammatory parameters (hsCRP; leucocytes). GI complaints were evaluated by a questionnaire. RESULTS: The intake of EcN resulted in a significantly lower increase in I-FABP and TBARS after exercise (p < 0.05). In contrast, no effect of EcN supplementation was found for hsCRP and leucocyte numbers. Similarly, no differences were found for levels of zonulin and claudin-3. Exercise-associated GI complaints were not affected by the probiotic supplement. CONCLUSION: The probiotic EcN reduced the exercise-associated increase in oxidative stress. This antioxidative mechanism probably leads to a reduction of GI epithelial damage after exhaustive exercise. The lack of EcN effects on other markers of GI permeability and systemic inflammation is most likely due to an inadequate exercise load, with rather small and insignificant exercise effects on these parameters.

Hepatic transcript profiling in early-lactation dairy cows fed rumen-protected niacin during the transition from late pregnancy to lactation.

In dairy cows, administration of high dosages of niacin (nicotinic acid, NA) was found to cause antilipolytic effects, which are mediated by the NA receptor hydroxyl-carboxylic acid receptor 2 (HCAR2) in white adipose tissue (WAT), and thereby an altered hepatic lipid metabolism. However, almost no attention has been paid to possible direct effects of NA in cattle liver, despite evidence that HCAR2 is also expressed in the liver and is even more abundant than in WAT. Because of this, we hypothesized that feeding a high dosage of rumen-protected NA to dairy cows influences critical metabolic or signaling pathways in the liver by inducing changes in the hepatic transcriptome. To identify these pathways, we applied genome-wide transcript profiling in liver biopsies obtained at d 7 postpartum (p.p.) from dairy cows used in our recent study; cows received either no NA (control group, n = 9) or 79 mg of rumen-protected NA/kg of body weight daily (NA group, n = 9) from 21 d before calving until 3 wk p.p. Hepatic transcript profiling revealed that 487 transcripts were differentially expressed (filter criteria: fold change >1.2 or <-1.2 and P < 0.05) in the liver at d 7 p.p. between cows fed NA and control cows. Substantially more transcripts were downregulated (n = 338), whereas only 149 transcripts were upregulated by NA in the liver of cows. Gene set enrichment analysis for the upregulated transcripts revealed that the most-enriched gene ontology biological process terms were exclusively related to immune processes, such as leukocyte differentiation, immune system process, activation of immune response, and acute inflammatory response. Gene set enrichment analysis of the downregulated transcripts showed that the most-enriched biological process terms were related to metabolic processes, such as cellular metabolic process, small molecule metabolic process, lipid catabolic process, organic cyclic compound metabolic process, small molecule biosynthetic process, and cellular lipid catabolic process. In conclusion, hepatic transcriptome analysis showed that rumen-protected NA induces genes that are involved mainly in immune processes, including acute phase response and stress response, in dairy cows at d 7 p.p. Thus, supplementation of a high dosage of rumen-protected NA to dairy cows in the periparturient period may induce or amplify the systemic inflammation-like condition that is typically observed in the liver of high-yielding dairy cows in the p.p. period.

Effect of endoplasmic reticulum stress on metabolic and stress signaling and kidney-specific functions in Madin-Darby bovine kidney cells.

Recent studies demonstrated induction of endoplasmic reticulum (ER) stress in tissues of cows after parturition, but knowledge about the effect of ER stress on important cellular processes, such as critical signaling and metabolic pathways, in cattle is scarce. Thus, the present study aimed to investigate the effect of ER stress induction on nuclear factor-κB (NF-κB), nuclear factor E2-related factor 2 (Nrf2), and sterol regulatory element-binding protein (SREBF1) pathway in Madin-Darby bovine kidney (MDBK) cells, a widely used in vitro model in ruminant research. To consider the kidney origin of MDBK cells, the effect on renal distal tubular cell-specific functions, such as transport processes and regulation of 1,25(OH)2D3 levels, was also studied. Treatment of MDBK cells with 2 different ER stress inducers, thapsigargin (TG) and tunicamycin (TM), strongly induced ER stress as evident from induction of ER stress target genes, increased phosphorylation of PKR-like ER kinase, and enhanced splicing of X-box binding protein 1. The TM decreased the protein concentration of NF-κB p50 and the mRNA levels of the NF-κB target genes. Likewise, TG decreased the mRNA concentration of tumor necrosis factor and tended to decrease NF-κB p50 protein and mRNA levels of NF-κB target genes. The mRNA levels of most of the Nrf2 target genes investigated were reduced by TG and TM in MDBK cells. Both ER stress inducers reduced the mRNA levels of SREBF1 and its target genes in MDBK cells. Interestingly, TG decreased, but TM increased the mRNA level of the Ca2+ binding protein calbindin 1, whereas the mRNA level of the plasma membrane Ca2+-transporting ATPase 1 remained unchained. The mRNA level of the cytochrome P450 component 24A1 involved in 1α-hydroxylation of 25(OH)D3 was strongly elevated, whereas the mRNA level of the cytochrome P450 component 27A1 catalyzing the breakdown of 1,25(OH)2D3 was markedly reduced by both ER stress inducers. The concentration of 1,25(OH)2D3 in the supernatant of MDBK cells was increased by approximately 15% by both TG and TM. The present study indicates that under conditions of ER stress, critical signaling pathways, such as NF-κB, Nrf2, and SREBF1, are inhibited, whereas the formation of 1,25(OH)2D3 is stimulated in bovine MDBK cells. Future studies are necessary to clarify the physiological relevance of these findings.

Potential of plant polyphenols to combat oxidative stress and inflammatory processes in farm animals.

Polyphenols are secondary plant metabolites which have been shown to exert antioxidative and antiinflamma tory effects in cell culture, rodent and human studies. Based on the fact that conditions of oxidative stress and inflammation are highly relevant in farm animals, polyphenols are considered as promising feed additives in the nutrition of farm animals. However, in contrast to many studies existing with model animals and humans, potential antioxidative and antiinflammatory effects of polyphenols have been less investigated in farm animals so far. This review aims to give an overview about potential antioxidative and antiinflammatory effects in farm animals. The first part of the review highlights the occurrence and the consequences of oxidative stress and inflammation on animal health and performance. The second part of the review deals with bioavailability and metabolism of polyphenols in farm animals. The third and main part of the review presents an overview of the findings from studies which investigated the effects of polyphenols of various plant sources in pigs, poultry and cattle, with particular consideration of effects on the antioxidant system and inflammation.

Sterol regulatory element-binding proteins are regulators of the sodium/iodide symporter in mammary epithelial cells.

The sodium/iodide symporter (NIS), which is essential for iodide concentration in the thyroid, is reported to be transcriptionally regulated by sterol regulatory element-binding proteins (SREBP) in rat FRTL-5 thyrocytes. The SREBP are strongly activated after parturition and throughout lactation in the mammary gland of cattle and are important for mammary epithelial cell synthesis of milk lipids. In this study, we tested the hypothesis that the NIS gene is regulated also by SREBP in mammary epithelial cells, in which NIS is functionally expressed during lactation. Regulation of NIS expression and iodide uptake was investigated by means of inhibition, silencing, and overexpression of SREBP and by reporter gene and DNA-binding assays. As a mammary epithelial cell model, the human MCF-7 cell line, a breast adenocarcinoma cell line, which shows inducible expression of NIS by all-trans retinoic acid (ATRA), and unlike bovine mammary epithelial cells, is widely used to investigate the regulation of mammary gland NIS and NIS-specific iodide uptake, was used. Inhibition of SREBP maturation by treatment with 25-hydroxycholesterol (5 µM) for 48h reduced ATRA (1 µM)-induced mRNA concentration of NIS and iodide uptake in MCF-7 cells by approximately 20%. Knockdown of SREBP-1c and SREBP-2 by RNA interference decreased the mRNA and protein concentration of NIS by 30 to 50% 48h after initiating knockdown, whereas overexpression of nuclear SREBP (nSREBP)-1c and nSREBP-2 increased the expression of NIS in MCF-7 cells by 45 to 60%, respectively, 48h after initiating overexpression. Reporter gene experiments with varying length of NIS promoter reporter constructs revealed that the NIS 5'-flanking region is activated by nSREBP-1c and nSREBP-2 approximately 1.5- and 4.5-fold, respectively, and activation involves a SREBP-binding motif (SRE) at -38 relative to the transcription start site of the NIS gene. Gel shift assays using oligonucleotides spanning either the wild-type or the mutated SRE at -38 of the NIS 5'-flanking region showed that in vitro-translated nSREBP-1c and nSREBP-2 bind only the wild-type but not the mutated SRE at -38 of NIS. Collectively, the present results from cell culture experiments with human mammary epithelial MCF-7 cells and from genetic studies show for the first time that the NIS gene and iodide uptake are regulated by SREBP in cultured human mammary epithelial cells. Future studies are necessary to clarify if the regulation of NIS expression and iodide uptake by SREBP also applies to the lactating bovine mammary epithelium.

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.

Molecular insights into the mechanisms of liver-associated diseases in early-lactating dairy cows: hypothetical role of endoplasmic reticulum stress.

The transition period represents the most critical period in the productive life of high-yielding dairy cows due to both metabolic and inflammatory stimuli, which challenge the liver and predispose dairy cows to develop liver-associated diseases such as fatty liver and ketosis. Despite the fact that all high-yielding dairy cows are affected by marked metabolic stress due to a severe negative energy balance (NEB) during early lactation, not all cows develop liver-associated diseases. Although the reason for this is largely unknown, this indicates that the capacity of the liver to cope with metabolic and inflammatory challenges varies between individual high-yielding dairy cows. Convincing evidence exists that endoplasmic reticulum (ER) stress plays a key role in the development of fatty liver, and it has been recently shown that ER stress occurs in the liver of high-yielding dairy cows. This indicates that ER stress may be involved in the development of liver-associated diseases in dairy cows. The present review shows that the liver of dairy cows during early lactation is exposed to several metabolic and inflammatory challenges, such as non-esterified fatty acids, tumour necrosis factor α, interleukin-1ß, reactive oxygen species and lipopolysaccharides, which are known inducers of ER stress. Thus, ER stress may represent a molecular basis for fatty liver development and account for the frequent occurrence of fatty liver and ketosis in high-yielding dairy cows. Interindividual differences between dairy cows in the activation of hepatic stress response pathways, such as nuclear factor E2-related factor 2, which is activated during ER stress and reduces the sensitivity of tissues to oxidative and inflammatory damage, might provide an explanation at the molecular level for differences in the capacity to cope with pathological inflammatory challenges during early lactation and the susceptibility to develop liver-associated diseases between early-lactating dairy cows with similar NEB and milk yield.

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.

Expression of fibroblast growth factor 21 in the liver of dairy cows in the transition period and during lactation.

Fibroblast growth factor 21 (FGF21) has been identified as a novel hormonal factor involved in the regulation of metabolic adaptations during energy deprivation. The present study aimed to investigate the expression of the FGF21 gene in the liver of dairy cows during the transition from pregnancy to lactation. Therefore, the relative mRNA abundance of FGF21 in liver biopsy samples of 20 dairy cows in late pregnancy (3 weeks pre-partum) and early lactation (1, 5, 14 weeks post-partum) was determined. It was observed that hepatic mRNA abundance of FGF21 at 1 week post-partum was dramatically increased (110-fold) compared to 3 weeks pre-partum (p < 0.001). With progress of lactation, mRNA concentration of FGF21 was declining; nevertheless, mRNA abundance at 5 and 14 weeks post-partum remained 25- and 10-fold increased compared to 3 weeks pre-partum (p < 0.001). Using a gene array technique, it was found that many genes involved in fatty acid oxidation, gluconeogenesis and ketogenesis were up-regulated during early lactation compared to late pregnancy. Moreover, there were positive linear correlations between hepatic mRNA concentration of FGF21 and mRNA concentrations of genes involved in ketogenesis as well as carnitine synthesis and carnitine uptake at various time-points during lactation, indicating that FGF21 could play a role in ketogenesis and carnitine metabolism in the liver of dairy cows (p < 0.05). In overall, the present study shows that expression of the FGF21 gene is strongly up-regulated during the transition period. It is assumed that the up-regulation of FGF21 might play an important role in the adaptation of liver metabolism during early lactation in dairy cows such as in other species.

Expression of target genes of nuclear factor E2-related factor 2 in the liver of dairy cows in the transition period and at different stages of lactation.

In the liver of dairy cows, the production of cytokines is enhanced during the periparturient phase, which in turn leads to inflammation and an impairment of hepatic function. Nuclear factor E2-related factor 2 (Nrf2) is a redox-sensitive transcription factor that controls the transcription of genes encoding various antioxidative and cytoprotective proteins. In the present study, we investigated the hypothesis that Nrf2 is activated in the liver of dairy cows during the periparturient phase to protect the liver against the deleterious effects of cytokines and reactive oxygen species. Therefore, we determined relative mRNA abundances of TNF (encoding tumor necrosis factor-α), various acute phase proteins and several Nrf2 target genes in liver biopsy samples of 20 dairy cows at each time point from 3 wk antepartum to 1, 5, and 14 wk postpartum. We observed an increase in mRNA abundances of TNF and acute-phase proteins [serum amyloid A 3 (SAA3), haptoglobin (HP), and C-reactive protein (CRP)] from 3 wk antepartum to 1 wk postpartum, indicative of a proinflammatory condition. Messenger RNA abundances of various Nrf2 target genes with antioxidative or cytoprotective functions [glutathione peroxidase 3 (GPX3); microsomal glutathione S-transferase 3 (MGST3); superoxide dismutase (SOD1); catalase (CAT); metallothioneins 1A, 1E, and 2A (MT1A, MT1E, and MT2A, respectively); NAD(P)H dehydrogenase, quinone 1 (NQO1); heme oxygenase 2 (HMOX2); and UDP glucuronosyltransferase 1 family, polypeptide A1 (UGT1A1)] were also greatly increased from 3 wk antepartum to 1 wk postpartum. From 1 wk postpartum to later lactation, mRNA abundances of all the Nrf2-target genes considered declined but remained at levels that were higher than those in 3 wk antepartum. No correlations were found, however, between plasma concentrations of nonesterified fatty acids or ß-hydroxybutyrate and mRNA abundances of Nrf2 target genes, indicating that a negative energy balance might not have been the main factor responsible for upregulation of those genes in the liver during early lactation. In conclusion, this study provides additional evidence that the periparturient phase in dairy cows is associated with a proinflammatory condition in the liver. Moreover, it is shown for the first time that the transition from pregnancy to lactation leads to a strong upregulation of Nrf2 target genes with antioxidative or cytoprotective properties, which might be another physiologic means to prevent the liver against damage by the inflammation process and an increased generation of reactive oxygen species.

Effects of fish oil and conjugated linoleic acids on carnitine homeostasis in laying hens.

1. The effects of n-3 polyunsaturated fatty acids (PUFA) and conjugated linoleic acids (CLA) on genes involved in carnitine homeostasis were compared in laying hens. Three groups of laying hens were fed on a control diet or a diet with either 3% of fish oil or CLA for 4 weeks. 2. Feed intake and egg production rate did not differ between the three groups. Diets with fish oil or CLA had only a weak effect on mRNA levels of PPARα target genes (ACO, CPT-I) in the liver and did not influence mRNA concentrations of the most important carnitine transporter OCTN2, enzymes of involved in carnitine synthesis (TMLD, TMABA-DH, BBD) or concentrations of carnitine in plasma, liver and total egg contents. 3. Hens fed the CLA diet had lower concentrations of free and total carnitine in egg yolk but higher concentrations of carnitine in albumen than control hens (P < 0·05), whereas the amount of free and total carnitine in whole egg did not differ. 4. In conclusion, the study showed that feeding fish oil or CLA causes only a weak activation of PPARα in tissues of laying hens that probably explained the lack of effect on carnitine homeostasis. The results contrast with those in humans and mice that show a significant effect of synthetic PPARα agonists on carnitine homeostasis in humans and mice.

Changes in the expression of hepatic genes involved in cholesterol homeostasis in dairy cows in the transition period and at different stages of lactation.

This study was performed to investigate changes in expression level of genes involved in hepatic cholesterol metabolism in the transition from pregnancy to lactation and during different stages of lactation in dairy cows. Therefore, relative mRNA abundances of several genes involved in various pathways of cholesterol homeostasis in liver biopsy samples of 20 dairy cows, taken in late pregnancy (3 wk prepartum) and early lactation (1, 5, and 14 wk postpartum), were determined. At 1 wk postpartum, hepatic mRNA abundances of genes involved in cholesterol synthesis (3-hydroxy-3-methylglutaryl-coenzyme A reductase, mevalonate kinase, and farnesyl diphosphate synthase), cholesterol uptake from blood (low-density lipoprotein receptor), bile acid synthesis (cholesterol-7α-hydroxylase), cholesterol efflux [ATP-binding cassette (ABC) transporter A1 and ABCG1], esterification of cholesterol (acyl-coenzyme A:cholesterol acyltransferase), and proteins involved in assembly and secretion of very low-density lipoproteins (microsomal triglyceride transfer protein, ApoB100) were increased compared with 3 wk prepartum. The mRNA abundances of most of these genes decreased after 1 wk of lactation and reached levels in 5 and 14 wk of lactation similar to those at 3 wk prepartum. Only mRNA abundances of cholesterol-7α-hydroxylase, ABC transporters, and ApoB100 remained at 5 and 14 wk postpartum at levels higher than those at 3 wk prepartum. Hepatic cholesterol abundance was highest at 1 wk postpartum and was, thereafter, decreasing to values similar to that at 3 wk prepartum. Overall, this study shows that the onset of lactation is associated with an increased expression of various genes involved in cholesterol metabolism in the liver of dairy cows, suggesting that pronounced changes in hepatic cholesterol metabolism take place in the periparturient phase.

Effects of a rumen-protected mixture of conjugated linoleic acids on hepatic expression of genes involved in lipid metabolism in dairy cows.

Supplementation of conjugated linoleic acids (CLA) reduces milk fat content in dairy cows and, thus, may be a useful dietary strategy to improve energy balance during early lactation. The present study was performed to investigate whether supplementation of CLA could have adverse effects on hepatic lipid metabolism such as observed in rodents. For this aim, 40 Holstein cows were allotted to 2 groups, which were fed daily 172 g of either a CLA-free, rumen-protected control fat (control group) or a rumen-protected CLA fat supplying 4.3g of cis-9,trans-11 CLA and 3.8 g of trans-10,cis-12 CLA per day (CLA group). To identify potential changes of lipid metabolism, expression of several genes involved in lipid metabolism was determined in liver biopsy samples taken at wk 5 of lactation, using a whole-genome gene chip. In the CLA group, milk fat content and daily milk fat yield were lower than in the control group. Milk yield was higher, whereas fat-corrected milk and energy-corrected milk were lower in the CLA group than in the control group. The CLA group, moreover, had an improved energy balance. To study potential effects of CLA on hepatic lipid metabolism, we considered 6 genes encoding fatty acid transporters, 7 genes involved in intracellular fatty acid transport, 21 and 7 genes, respectively, involved in mitochondrial and peroxisomal ß-oxidation, 6 genes of carnitine metabolism, 3 genes of ketogenesis, 21 genes involved in fatty acid and triacylglycerol synthesis, 17 genes involved in cholesterol metabolism, and 20 genes involved in lipoprotein metabolism. None of these genes was differentially regulated between the CLA group and the control group. Gene chip data were confirmed by quantitative PCR analysis, which revealed no difference in the expression of key enzymes of various pathways such as lipogenesis, ß-oxidation, and ketogenesis between the 2 groups of cows. In line with those findings, concentrations of triacylglycerols and cholesterol in liver and plasma were not different between the 2 groups of cows. In conclusion, the present study shows that CLA supplementation at a dose effective for milk fat depression does not induce adverse effects on hepatic lipid metabolism in dairy cows.

Influence of a rumen-protected conjugated linoleic acid mixture on carcass traits and meat quality in young Simmental heifers.

The aim of the present study was to investigate the influence of feeding rumen-protected CLA during the early growing period on physical and chemical beef properties in young Simmental heifers. A total of 36 heifers (5 mo old; initial BW 185 ± 21 kg) were fed 250 g of different rumen-protected fats daily for 16 wk in 1 of 3 treatment groups: 250 g of a CLA-free control fat; 100 g of a CLA fat containing 2.4% of cis-9,trans-11 CLA and 2.1% of trans-10,cis-12 CLA and 150 g control fat; or 250 g of the CLA fat. Heifer growth performance variables as well as carcass weight, classification (conformation and fatness), and weights of organs and fat depots were not affected (P > 0.05) by CLA supplementation. Concentration of trans-10,cis-12 CLA in tissues (LM and subcutaneous fat) was dose-dependently increased (P < 0.01) by CLA supplementation, whereas that of cis-9,trans-11 CLA in these tissues did not differ (P > 0.05) between groups. The ratio of SFA to MUFA was increased (P < 0.01) in tissues of CLA-fed heifers compared with control heifers. Concentration of α-tocopherol in LM was greater (P = 0.01) in heifers of the 2 CLA groups than in control heifers. Other quality characteristics such as drip loss during storage, cooking loss, intramuscular fat content, and color variables in LM did not differ (P > 0.05) between groups. In conclusion, the present study demonstrates that feeding rumen-protected CLA during the early growing period changes tissue fatty acid composition but does not influence beef quality variables. Performance variables and carcass traits in young heifers, unlike in pigs and laboratory animals, are not influenced by CLA feeding.

Supplementation with l-carnitine downregulates genes of the ubiquitin proteasome system in the skeletal muscle and liver of piglets.

Supplementation of carnitine has been shown to improve performance characteristics such as protein accretion in growing pigs. The molecular mechanisms underlying this phenomenon are largely unknown. Based on recent results from DNA microchip analysis, we hypothesized that carnitine supplementation leads to a downregulation of genes of the ubiquitin proteasome system (UPS). The UPS is the most important system for protein breakdown in tissues, which in turn could be an explanation for increased protein accretion. To test this hypothesis, we fed sixteen male, four-week-old piglets either a control diet or the same diet supplemented with carnitine and determined the expression of several genes involved in the UPS in the liver and skeletal muscle. To further determine whether the effects of carnitine on the expression of genes of the UPS are mediated directly or indirectly, we also investigated the effect of carnitine on the expression of genes of the UPS in cultured C2C12 myotubes and HepG2 liver cells. In the liver of piglets fed the carnitine-supplemented diet, the relative mRNA levels of atrogin-1, E214k and Psma1 were lower than in those of the control piglets (P < 0.05). In skeletal muscle, the relative mRNA levels of atrogin-1, MuRF1, E214k, Psma1 and ubiquitin were lower in piglets fed the carnitine-supplemented diet than that in control piglets (P < 0.05). Incubating C2C12 myotubes and HepG2 liver cells with increasing concentrations of carnitine had no effect on basal and/or hydrocortisone-stimulated mRNA levels of genes of the UPS. In conclusion, this study shows that dietary carnitine decreases the transcript levels of several genes involved in the UPS in skeletal muscle and liver of piglets, whereas carnitine has no effect on the transcript levels of these genes in cultivated HepG2 liver cells and C2C12 myotubes. These data suggest that the inhibitory effect of carnitine on the expression of genes of the UPS is mediated indirectly, probably via modulating the release of inhibitors of the UPS such as IGF-1. The inhibitory effect of carnitine on the expression of genes of the UPS might explain, at least partially, the increased protein accretion in piglets supplemented with carnitine.

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-1ß, 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.

Effect of a rumen-protected conjugated linoleic acid mixture on hepatic lipid metabolism in heifers.

This study was performed to assess the effects of rumen-protected conjugated linoleic acid (CLA) on hepatic lipid metabolism in heifers. In particular, it was of interest whether feeding CLA causes development of fatty liver as observed recently in mice. Thirty-six growing heifers with an initial body weight of 185 kg were allotted to three treatment groups and fed daily 250 g of different rumen-protected fats for 16 weeks: The control group received 250 g of a CLA-free control fat, the CLA100 group received 100 g of a CLA fat containing 2.4% of cis-9, trans-11 CLA and 2.1% of trans-10, cis-12 CLA and 150 g control fat and the CLA250 group received 250 g of the CLA fat. CLA supplementation had no effect on animal performance parameters, liver weight and hepatic triglyceride concentration. Moreover, mRNA expression of hepatic genes involved in lipogenesis, ß-oxidation and fatty acid transport was not influenced by dietary CLA. The fatty acid composition of hepatic total lipids, with particular consideration of ratios of fatty acids indicative of Δ9-, Δ6- and Δ5-desaturation, was also less influenced by dietary CLA. In conclusion, the study shows that dietary rumen-protected CLA has less effect on hepatic lipid metabolism in young heifers and does not induce the development of a fatty liver such as in mice.

Short communication: Dietary conjugated linoleic acid down-regulates fatty acid transporters in the mammary glands of lactating rats.

Recent studies indicated that reduction of milk triacylglycerol concentrations by dietary conjugated linoleic acid (CLA) involves an impairment of both de novo fatty acid synthesis and uptake of fatty acids from circulating triacylglycerol-rich lipoproteins into the mammary gland. However, nonesterified fatty acids (NEFA) in the plasma released from adipose tissue and taken up into the mammary gland by fatty acid transporters are a further important source of fatty acids available for milk triacylglycerol synthesis. Therefore, the aim of the present study was to investigate the effect of dietary CLA on plasma concentrations of NEFA and the expression of fatty acid transporters in the mammary glands of lactating rats fed either a CLA diet or a control diet. Dams fed diets with CLA had a greater concentration of NEFA in plasma than those fed the control diet. In addition, relative mRNA concentrations of fatty acid transporters (fatty acid translocase/CD36, fatty acid transport protein, and plasma membrane fatty acid binding protein) were about 45, 75, and 70% lower, respectively, in the mammary gland of dams fed diets with CLA compared with those fed the control diet. In conclusion, the present findings indicate that reduced uptake of circulating NEFA released from white adipose tissue into the mammary gland could also contribute to the reduction of milk triacylglycerol concentrations by dietary CLA in rats. The mechanism through which CLA inhibits expression of fatty acid transporters deserves further study.

Oxidized fat induces oxidative stress but has no effect on NF-kappaB-mediated proinflammatory gene transcription in porcine intestinal epithelial cells.

OBJECTIVE AND DESIGN: The effect of oxidized frying oils on PPARgamma which is a potent inhibitor of inflammatory responses in the intestine is currently unknown. Thus, the present study aimed to explore the effect of oxidized frying oil on PPARgamma DNA-binding and proinflammatory responses in intestinal epithelial cells. MATERIAL AND METHODS: 18 male pigs were fed two different diets containing either fresh fat or oxidized fat (n = 9 each). After 28 d, intestinal epithelial cells were isolated and analyzed for PPARgamma DNA-binding, NF-kappaB DNA-binding and NF-kappaB target gene expression. In addition, markers of lipid peroxidation and antioxidant status were determined. RESULTS: Feeding the oxidized fat slightly, but not significantly, activated PPARgamma DNA-binding in intestinal epithelial cells when compared to fresh fat. In addition, oxidized fat increased the concentration of TBARS and reduced the concentrations of alpha-tocopherol and activities of antioxidant enzymes relative to fresh fat (P < 0.05). No effect of the oxidized fat was observed on NF-kappaB DNA-binding and NF-kappaB target gene expression in intestinal epithelial cells. CONCLUSIONS: The present study suggests that moderate PPARgamma activation and induction of oxidative stress by oxidized frying oil have no implication for NF-kappaB-mediated proinflammatory gene expression in porcine intestinal epithelial cells.

Peptides and hydrolysates from casein and soy protein modulate the release of vasoactive substances from human aortic endothelial cells.

Food proteins were shown to affect atherogenic risk factors, which is supposed to be related to specific peptide sequences encrypted within their primary sequence. The aim of this study was to evaluate the effects of peptides and hydrolysates from two food proteins, casein and soy protein, on endothelial cell functions (cell proliferation and release of vasoactive substances). Cell proliferation was not influenced by dipeptides and most of the tripeptides, whereas several total hydrolysates from casein and soy protein inhibited cell proliferation at higher concentrations (>0.25 mg/mL; P<0.05). The release of one or more of the vasoactive substances, thromboxan B2 (stable marker of thromboxan A2), 6-keto-prostaglandin F1alpha (stable marker of prostaglandin I2), endothelin-1, and nitric oxide, was significantly influenced by the incubation with various peptides compared with control cells (P<0.05). Various hydrolysate fractions from casein and soy protein influenced the release of 6-keto-prostaglandin F1alpha and nitric oxide (P<0.05) but did not influence the release of thromboxan B2 and endothelin-1. In conclusion, the present study demonstrates that peptides and hydrolysate fractions from casein and soy protein influence endothelial cell function as evidenced by the modulation of endothelial cell proliferation and alterations in the release of vasoactive substances.