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This study investigated the hypothesis that dietary supplementation of leucine (Leu) above actual recommendations activates protein synthesis and inhibits protein degradation pathways on the molecular level and supports higher muscle growth in broilers. Day-old male Cobb-500 broilers (n = 180) were allotted to 3 groups and phase-fed 3 different corn-wheat-soybean meal-based basal diets during periods 1 to 10, 11 to 21, and 22 to 35 D. The control group (L0) received the basal diet which met the broiler's requirements of nutrients and amino acids for maintenance and growth. Groups L1 and L2 received basal diets supplemented with Leu to exceed recommendations by 35 and 60%, respectively, and isoleucine (Ile) and valine (Val) were supplemented to keep Leu: Ile and Leu: Val ratios fixed. Samples of liver and breast muscle and pancreas were collected on days 10, 21, and 35. The gene expression and abundance of total and phosphorylated proteins involved in the mammalian target of rapamycin pathway of protein synthesis, in the ubiquitin-proteasome pathway and autophagy-lysosomal pathway of protein degradation, in the general control nonderepressible 2/eukaryotic translation initiation factor 2A pathway involved in the inhibition of protein synthesis, and in the myostatin-Smad2/3 pathway involved in myogenesis were evaluated in the muscle, as well as expression of genes involved in the growth hormone axis. Growth performance, feed intake, the feed conversion ratio, and carcass weights did not differ between the 3 groups (P > 0.05). Plasma concentrations of Leu, Ile, and Val and of their keto acids, and the activity of the branched-chain α-keto acid dehydrogenase in the pancreas increased dose dependently with increasing dietary Leu concentrations. In the breast muscle, relative mRNA abundances of genes and phosphorylation of selected proteins involved in all investigated pathways were largely uninfluenced by dietary Leu supplementation (P > 0.05). In summary, these data indicate that excess dietary Leu concentrations do not influence protein synthesis or degradation pathways, and subsequently do not increase muscle growth in broilers at fixed ratios to Ile and Val.

BACKGROUND Specific dietary proteins exert strong health-related effects compared with casein. OBJECTIVE Herein, the hypothesis was tested using screening and conventional biochemical and molecular biological techniques that protein-rich insect meal compared with casein influences metabolic health in hyperlipidemic rats. METHODS A 4-wk feeding trial with male, 8-wk-old homozygous obese Zucker rats (n = 36) and male, 8-wk-old heterozygous lean Zucker rats (n = 12) was performed. Obese rats were randomly divided into 3 obese groups (OC, OI50, and OI100) of 12 rats each and lean rats served as a lean control group (LC). LC and OC were fed a control diet with 20% casein as protein source, whereas in OI50 and OI100 50% and 100% of the casein, respectively, was replaced isonitrogenously by insect meal from Tenebrio molitor L. All data were analyzed by 1-factor ANOVA, except transcriptomic data which were analyzed by groupwise comparisons with the OC group. RESULTS Transcript profiling revealed a coordinated inhibition by -17% to -521% and -37% to -859% of genes involved in fatty acid, triacylglycerol (TG), and cholesterol biosynthesis in the livers of OI100 and OI50, respectively, compared with OC (P

Hepatic steatosis is a very common response to liver injury and often attributed to metabolic disorders. Prior studies have demonstrated the efficacy of a biotechnologically produced oyster mushroom (Pleurotus sajor-caju, PSC) in alleviating hepatic steatosis in obese Zucker rats. This study aims to elucidate molecular events underlying the anti-steatotic effects of PSC. Tandem mass tag (TMT) peptide labeling coupled with LC-MS/MS/MS was used to quantify and compare proteins in the livers of lean Zucker rats fed a control diet (LC), obese Zucker rats fed the same control diet (OC) and obese Zucker rats fed the control diet supplemented with 5% PSC (OPSC) for 4 weeks. Using this technique 3128 proteins could be quantified, out of which 108 were differentially abundant between the OPSC and OC group. Functional enrichment analysis of the up-regulated proteins showed that these proteins were mainly involved in metabolic processes, while the down-regulated proteins were involved in inflammatory processes. Results from proteomic analysis were successfully validated for two up-regulated (carbonic anhydrase 3, regucalcin) and two down-regulated (cadherin-17, ceruloplasmin) proteins by means of immunoblotting. Significance Valorization of low-grade agricultural waste by edible fungi, such as the mushroom Pleurotus sajor-caju (PSC), represents a promising strategy for the production of protein rich biomass since they boast of a unique enzyme system that has the ability to recover nutrients and energy from biodegradable waste. Herein, we describe the metabolic effects of PSC feeding using a combined quantitative proteomics and bioinformatics approach. In total, 108 proteins were identified to be regulated by PSC feeding in the liver of the obese rats. Complementary usage of a bioinformatics approach allowed us to decipher the mechanisms underlying the recently observed lipid-lowering and anti-inflammatory activity of PSC feeding in obese Zucker rats, namely a reduction of fatty acid synthesis, an improvement of hepatoprotective mechanisms and an enhancement of anti-inflammatory effects.

The genes encoding sodium/iodide symporter (NIS) and thyroid peroxidase (TPO), both of which are essential for thyroid hormone (TH) synthesis, were shown to be regulated by sterol regulatory element-binding proteins (SREBP)-1c and -2. In the present study we tested the hypothesis that transcription of a further gene essential for TH synthesis, the thyroglobulin (TG) gene, is under the control of SREBP. To test this hypothesis, we studied the influence of inhibition of SREBP maturation and SREBP knockdown on TG expression in FRTL-5 thyrocytes and explored transcriptional regulation of the TG promoter by reporter gene experiments in FRTL-5 and HepG2 cells, gel shift assays and chromatin immunoprecipitation. Inhibition of SREBP maturation by 25-hydroxycholesterol and siRNA-mediated knockdown of either SREBP-1c or SREBP-2 decreased mRNA and protein levels of TG in FRTL-5 thyrocytes. Reporter gene assays with wild-type and mutated TG promoter reporter truncation constructs revealed that the rat TG promoter is transcriptionally activated by nSREBP-1c and nSREBP-2. DNA-binding assays and chromatin immunoprecipitation assays showed that both nSREBP-1c and nSREBP-2 bind to a SREBP binding motif with characteristics of an E-box SRE at position -63 in the rat TG promoter. In connection with recent findings that NIS and TPO are regulated by SREBP in thyrocytes the present findings support the view that SREBP are regulators of essential steps of TH synthesis in the thyroid gland such as iodide uptake, iodide oxidation and iodination of tyrosyl residues of TG. This moreover suggests that SREBP may be molecular targets for pharmacological modulation of TH synthesis.

This study investigated the hypothesis that dietary fats rich in lauric (C12) and myristic acid (C14) increase broiler performance and that the underlying mechanism involves antimicrobial effects on gut bacteria and changes in gut morphology. One hundred eighty 1-day-old Cobb-500 broilers were allotted to 3 groups. All groups received a basal diet consisting of maize, wheat, soybean meal, and a fat source (4.5, 7.0, 7.6, and 8.0% of fat product in the diet during d 1 to 9, 10 to 17, 18 to 27, and 28 to 35, respectively) until 35 d of age. The diet of the control group contained a fat with 67% of oleic and linoleic acid and 1.4% of C12 and C14 of total fatty acids, that of the esterified lauric and myristic acid (ELA) group a fat with 33% of esterified C12 and C14 and that of the free lauric and myristic acid (FLA) group a fat with 31% of both esterified and free (1:1) C12 and C14 (6 replicates/treatment, 10 birds/replicate). Gain and feed consumption did not differ between groups, but feed:gain was lower in FLA group as compared to the control group (P < 0.05). Carcass weight, liver weight, triglyceride content of liver and muscle, and muscle cholesterol were similar between groups; however, breast muscle weight was higher in the FLA than in the control group (P < 0.05). The villus height:crypt depth ratio of the duodenal wall did not differ between groups, but in the jejunum, it was lower in the FLA group as compared to the control group (P < 0.05). DNA copy numbers of Lactobacillus, Bifidobacteria, Enterobacteria, Escherichia coli, and Campylobacter jejuni in jejunal digesta were similar among groups. The study shows that dietary fats rich in free C12 and C14 improved feed:gain and breast muscle yield, but the observed effects could not be conclusively explained based on the parameters measured. The decreased jejunal villi:crypt ratio may point to changes in gut protein or cell turnover.

Objective To evaluate medical efficacy and safety of crestal, minimally invasive sinus floor augmentation (MISFA) using an innovative method based on high hydraulic pressure. Study design Twenty MISFA using the novel Jeder-System were performed in 18 patients at 2 study sites in Vienna, Austria. The Jeder-System consists of the Jeder-drill, the Jeder-pump, and a connecting tube-set. The pump generates high hydraulic pressure (1.5 bar) pushing back the sinus membrane from the drill at the first perforation. The pump also monitors the whole procedure by constantly measuring pressure and volume. Results Five percent membrane perforation rate (1/20) only detected in the postoperative computed tomography scan and without implication for implant placement. Height gain of 9.2 ± 1.7 mm achieved (from 4.6 ± 1.4 mm to 13.8 ± 2.3 mm). Average patient satisfaction was 9.82 on scale from 1 to 10 (10 = very satisfied). Mean duration of sick leave was 0.19 days. 18-month survival rate was 95% (1/20 implant lost). Conclusions Within the limits of a prospective open cohort study with 20 cases, our data demonstrate the safety and medical efficacy of the novel method.

In the present study, we tested the hypothesis that niacin increases the oxidative capacity of muscle by increasing the oxidative type I muscle fiber content. Twenty-four obese Zucker rats were assigned to 2 groups of 12 rats that were fed either a control diet (O group) or a diet supplemented with 750 mg/kg diet niacin (O+N group) for 4 wk. In addition, one group of lean rats (L group) was included in the experiment and fed the control diet for 4 wk. Plasma and liver concentrations of TG were markedly greater in obese groups than in the L group but markedly lower in the O+N group than in the O group (P < 0.05). Rats of the O+N group had a higher percentage of oxidative type I fibers and higher mRNA levels of genes encoding regulators of muscle fiber composition (Ppard, Ppargc1a, Ppargc1b), angiogenic factors (Vegfa, Vegfb), and genes involved in fatty acid utilization (Cpt1b, Slc25a20, Slc22a4, Slc22a5, Slc27a1) and oxidative phosphorylation (Cox4i1, Cox6a2) and a higher activity of the mitochondrial oxidative enzyme succinate dehydrogenase in muscle than rats of the O and L groups (P < 0.05). These niacin-induced changes in muscle metabolic phenotype are indicative of an increased capacity of muscle for oxidative utilization of fatty acids and are likely mediated by the upregulation of Ppard, Ppargc1a, and Ppargc1b, which are key regulators of muscle fiber composition, mitochondrial biogenesis, angiogenesis, and genes involved in fatty acid catabolism and oxidative phosphorylation. The increased utilization of fatty acids by muscle might contribute to the strong TG-lowering effect of niacin treatment.

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.

The present study aimed to explore the lactation-induced changes in hepatic gene expression in sows (Sus scrofa) during lactation. Using a porcine whole-genome microarray a total of 632 differentially expressed genes in the liver of lactating compared to non-lactating sows could be identified. Enrichment analysis revealed that the differentially expressed genes were mainly involved in fatty acid metabolism, pyruvate metabolism, glutathione metabolism, glycine, serine and threonine metabolism, citrate cycle, glycerophospholipid metabolism, PPAR signaling, and focal adhesion. The most striking observation with respect to intermediary metabolism was that genes involved in fatty acid catabolism, the catabolism of gluconeogenic amino acids, the citrate cycle and the respiratory chain were up-regulated in the liver of sows during lactation. With respect to immune response, it could be demonstrated that genes encoding acute phase proteins and genes involved in tissue repair were up-regulated and genes encoding adhesion molecules were down-regulated in the liver of sows during lactation. The results indicate that energy-generating pathways and pathways involved in the delivery of gluconeogenic substrates are induced in sow liver during lactation. The alterations of expression of genes encoding proteins involved in immune response suggest that lactation in sows may cause an adaptive immune response that possibly counteracts hepatic inflammation.

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.

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.

Convincing evidence from studies with peroxisome proliferator-activated receptor (PPAR)α-deficient mice suggested that the carnitine biosynthetic enzyme γ-butyrobetaine dioxygenase (BBD) is regulated by PPARα. However, the identification of BBD as a direct PPARα target gene as well as its exact regulation remained to be demonstrated. In silico-analysis of the mouse BBD promoter revealed seven putative peroxisome proliferator response elements (PPRE) with high similarity to the consensus PPRE. Luciferase reporter gene assays using mutated and non-mutated serial 5′-truncation BBD promoter reporter constructs revealed that one PPRE located at −75 to −87 relative to the transcription start site in the proximal BBD promoter is probably functional. Using gel shift assays we observed in vitro-binding of PPARα/RXRα heterodimer to this PPRE confirming that it is functional. In conclusion, the present study clearly shows that mouse BBD is a direct PPARα target gene and that transcriptional up-regulation of mouse BBD by PPARα is likely mediated by binding of the PPARα/RXR heterodimer to one PPRE located in its proximal promoter region. The results confirm emerging evidence from recent studies that PPARα plays a key role in the regulation of carnitine homeostasis by controlling genes involved in both, carnitine synthesis and carnitine uptake.

In rodents, fasting increases the carnitine concentration in the liver by an up-regulation of enzymes of hepatic carnitine synthesis and novel organic cation transporter (OCTN) 2, mediated by activation of peroxisome proliferator-activated receptor (PPAR) alpha. This study was performed to investigate whether such effects occur also in pigs which like humans, as nonproliferating species, have a lower expression of PPARalpha and are less responsive to treatment with PPARalpha agonists than rodents. An experiment with 20 pigs was performed, which were either fed a diet ad-libitum or fasted for 24 h. Fasted pigs had higher relative mRNA concentrations of the PPARalpha target genes carnitine palmitoyltransferase 1 and acyl-CoA oxidase in liver, heart, kidney, and small intestinal mucosa than control pigs, indicative of PPARalpha activation in these tissues (P

Hepatic PPARalpha acts as the primary mediator of the adaptive response to fasting by upregulation of a number of genes involved in fatty acid catabolism. Whether carnitine-acylcarnitine translocase (CACT), which mediates the import of acylcarnitines into the mitochondrial matrix for subsequent beta-oxidation of fatty acid moieties, is also regulated by PPARalpha in the liver has not yet been investigated.Herein, we observed that hepatic mRNA abundance of CACT was increased by both, fasting and treatment with PPARalpha agonist WY-14,643 in wild-type mice but not PPARalpha-knockout mice (P0.05). Cell culture experiments revealed that CACT mRNA abundance was higher in liver cells treated with either WY-14,643 or PPARdelta agonist GW0742, but not with PPARgamma agonist troglitazone (TGZ) than in control cells (P0.05). In addition, reporter assays revealed activation of mouse CACT promoter by WY-14,643 and GW0742, but not TGZ. Moreover, deletion and mutation analyses of CACT promoter and 5'-UTR revealed one functional PPRE in the 5'-UTR of mouse CACT.CACT is upregulated by PPARalpha and PPARdelta, probably by binding to a functional PPRE at position +45 to +57 relative to the transcription start site. The upregulation of CACT by PPARalpha and PPARdelta, which are both important for the regulation of fatty acid oxidation in tissues during fasting, may increase the import of acylcarnitine into the mitochondrial matrix during fasting.

In contrast to other species, less is known about carnitine homeostasis in the pig. This study was performed to yield information about the site of carnitine synthesis and carnitine concentrations in various tissues of pigs (Sus scrofa). We found that among several pig tissues, a considerable activity of gamma-butyrobetaine dioxygenase (BBD), the last enzyme of carnitine synthesis, exists, like in humans and several other species, only in liver and kidney. Activity of that enzyme in liver and kidney was lower at birth than in the subsequent weeks of life. Highest carnitine concentrations were found in skeletal muscle and heart. Carnitine concentrations in plasma, liver and kidney at birth were higher than in the subsequent weeks of life in spite of the low BBD activity at birth. In conclusion, this study shows that liver and kidney are the major sites of carnitine synthesis and that neonatal pigs do not have an insufficient carnitine status.

For the investigation of the metabolism and biosynthesis of carnitine, sensitive determination of carnitine and its metabolic precursors, trimethyllysine and gamma-butyrobetaine, is required. We present here a new simplified method for the analysis of carnitine, its acetyl- and propyl esters, as well as trimethyllysine and gamma-butyrobetaine without need for derivatization reactions by means of normal-phase LC and electrospray ionization tandem mass spectrometry. The limits of quantification were between 5 nM for acetyl carnitine and 70 nM for carnitine. Relative standard deviations in a fivefold determination of standard solutions were between

Protein tyrosine phosphatase 1B (PTP1B) is a key enzyme in the counter-regulation of insulin signaling and in the stimulation of fatty acid synthesis. Selenium (Se), via the activities of glutathione peroxidase (GPx) and thioredoxin reductase (TrxR), is involved in the removal of H2O2 and organic peroxides, which are critical compounds in the modulation of PTP1B activity via glutathionylation. Our study with growing rats investigated how the manipulation of dietary Se concentration influences the regulation of PTP1B and lipogenic effects mediated by PTP1B. Weanling albino rats were divided into 3 groups of 10. The negative control group (NC) was fed a Se-deficient diet for 8 wk. Rats in groups Se75 and Se150 received diets supplemented with 75 or 150 mg Se/kg. Se supplementation of the rats strongly influenced expression and activity of the selenoenzymes cytosolic GPx, plasma GPx, phospholipidhydroperoxide GPx, and cytosolic TrxR, and liver PTP1B. Liver PTP1B activity was significantly higher in groups Se75 and Se150 than in the NC group and this was attributed to a lowered inhibition of the enzyme by glutathionylation. The increased liver PTP1B activity in groups Se75 and Se150 resulted in 1.1- and 1.4-fold higher liver triglyceride concentrations than in the NC rats. The upregulation of the sterol regulatory element binding protein-1c and of fatty acid synthase, 2 PTP1B targets, provided a possible explanation for the lipogenic effect of PTP1B due to the manipulation of dietary Se. We therefore conclude that redoxregulated proteins, such as PTP1B, represent important interfaces between dietary antioxidants such as Se and the regulation of metabolic processes. J. Nutr. 138: 2328‐2336, 2008.

Activation of PPARalpha by clofibrate has recently been shown to cause upregulation of the high-affinity carnitine transporter novel organic cation transporter (OCTN) 2 in small intestine. This strongly suggests that PPARalpha activation in response to clofibrate treatment improves the absorption of carnitine from the diet. To test this hypothesis, we performed an experiment with rats which were fed diets with or without 5 g clofibrate/kg diet and with or without 5 g L-carnitine/kg diet. PPARalpha was significantly activated by clofibrate in small intestine as evidenced by increased relative mRNA concentrations of the PPARalpha target gene acyl-CoA oxidase (P0.05). Relative mRNA concentration of OCTN2 in small intestine was significantly increased by clofibrate (P0.05) but not the carnitine supplementation, whereas relative mRNA concentrations of other carnitine transporters (OCTN1, ATB(0+)) in small intestine were not influenced by either clofibrate or carnitine. The absorption rate of carnitine in small intestine was markedly higher in rats treated with clofibrate than in those treated without clofibrate (P0.05). In conclusion, the present study shows that administration of clofibrate to rats increases carnitine absorption in small intestine which is probably due to the observed upregulation of OCTN2 mediated by activation of PPARalpha.

Recent studies have shown that treatment of rodents with agonists of peroxisome proliferator-activated receptor (PPAR)-alpha causes an up-regulation of novel organic cation transporter (OCTN)-2, a carnitine transporter, and increases carnitine concentration in the liver. This study was performed to investigate whether such effects occur also in pigs which like humans have a lower expression of PPAR alpha and are less responsive to treatment with PPAR alpha agonists than rodents. An experiment with 18 pigs was performed which were fed a control diet or the same diet supplemented with 5 g clofibrate/kg for 28 days. Pigs treated with clofibrate had higher relative mRNA concentrations of OCTN2 in liver (3.1-fold), skeletal muscle (1.5-fold) and epithelial cells from small intestine (1.8-fold) than control pigs (P

Recent studies have shown that l -carnitine supplementation of sows during pregnancy and lactation increases their reproductive performance, but the optimum supplementation level has not yet been determined. In this study we investigated the effects of supplementation of 25 and 50 mg l -carnitine/kg on traits of reproductive performance of sows in production stocks. A trial with 2118 sows was conducted which were assigned to three groups, fed either control diets with low native l -carnitine concentrations or the same diets supplemented with 25 or 50 mg of l -carnitine/kg during pregnancy and lactation. Sows supplemented with 25 or 50 mg l -carnitine/kg diet had more piglets born alive than control sows ( P l -carnitine/kg were also heavier at birth and on day 21 than those of control sows ( P l -carnitine/kg diet and those supplemented with 25 mg l -carnitine/kg did not differ in the number of piglets born alive or piglet and litter weights at birth. On day 21, however, weights of piglets and litters were higher in sows supplemented with 50 mg l -carnitine/kg diet than in sows supplemented with 25 mg l -carnitine/kg diet ( P l -carnitine/kg diet has beneficial effects on the reproductive performance of sows. Supplementation at 50 mg l -carnitine/kg diet is, however, superior with respect to weight development of litters during the suckling period.

Feeding oxidized fats to lactating rats causes a strong reduction of triacylglycerol concentration in the milk. The reason for this, however, has not yet been elucidated. Pregnant Sprague-Dawley rats were assigned to 2 groups of 11 rats each and fed diets containing either fresh fat (FF group) or an oxidized fat (OF group) from d 1 to d 20 of lactation. Concentrations of triacylglycerols and long-chain fatty acids in the milk and weight gain of suckling pups were lower in the OF group than in the FF group (P < 0.05). Concentrations of medium-chain fatty acids in the milk and messenger RNA (mRNA) abundance of lipogenic enzymes in the mammary gland did not differ between the 2 groups of rats. However, the OF group had a lower concentration of triacylglycerols and nonesterified fatty acids (NEFA) in plasma and lower mRNA concentrations of lipoprotein lipase and fatty acid transporters in the mammary gland than the FF group (P < 0.05). Moreover, the OF group had higher mRNA concentrations of hepatic lipase, fatty acid transporters, and several genes involved in fatty acid oxidation in the liver than the FF group (P < 0.05). The present findings suggest that a dietary oxidized fat lowers the concentration of triacylglycerols in the milk by a reduced uptake of fatty acids from triacylglycerol rich-lipoproteins and NEFA into the mammary gland. The study, moreover, indicates that an oxidized fat impairs normal metabolic adaptations during lactation, which promote the utilization of metabolic substrates by the mammary gland for the synthesis of milk.

Activation of PPARalpha by clofibrate has recently been shown to cause upregulation of carnitine transporter organic cation transporter (OCTN) 2 and elevated carnitine concentrations in rat liver. The present study has been conducted to further explore the effect of clofibrate on OCTN expression, carnitine biosynthesis, and carnitine accumulation in different rat tissues, and thus two groups of rats were fed diets containing 0.5% clofibrate or 0% clofibrate (control group). PPARalpha-responsive genes were markedly upregulated in the liver (P

The present study investigated whether treatment of female rats with pivalate affects their reproductive function. Therefore, two experiments with female rats were performed. The first experiment included two groups of rats which received drinking water without (control) or with 20 mmol pivalate/L. The second experiment included a control group (which received drinking water without pivalate and a diet without added carnitine) and four groups which received drinking water with 20 mmol/L pivalate and diets without or with 1, 3 or 5 g added carnitine/kg, respectively. In both experiments, rats treated with pivalate had a lower number of pups born alive and, as a consequence of this, lower litter weights than control rats (p0.05); pup weights were not altered by pivalate treatment. Supplementation of dietary carnitine in Experiment 2 increased plasma and tissue carnitine concentration even in excess of those in control rats but did not restore normal litter sizes. This study shows for the first time that pivalate affects the reproductive function in female rats independent of its effect on the carnitine status.

Soy intake acts hypolipidemically. Besides isoflavones, soy protein itself is suggested to influence plasma lipid concentrations. We investigated the effects of an alcohol-washed isoflavone-poor soy protein isolate on plasma and liver lipids and the hepatic expression of genes encoding proteins involved in cholesterol and fatty acid metabolism. Therefore, rats were fed diets containing 200 g/kg of either ethanol-extracted soy protein isolate or casein over 22 days. Rats fed soy protein isolate had markedly lower concentrations of liver cholesterol and lower concentrations of triglycerides in the liver and in plasma than rats fed casein (P.05). Rats fed soy protein isolate had lower relative mRNA concentrations of sterol-regulatory element-binding protein (SREBP)-2, 3-hydroxy-3-methylglutaryl coenzyme A reductase, low-density lipoprotein receptor, cholesterol 7alpha-hydroxylase, apolipoprotein B, Delta9-desaturase and glucose-6-phosphate dehydrogenase in the liver than rats fed casein (P.05). Hepatic mRNA concentration of SREBP-1c tended to be lower in rats fed soy protein isolate (P.10). Hepatic mRNA concentrations of insulin-induced gene (Insig) 1 and Insig-2 and of microsomal triglyceride transfer protein, as well as plasma concentrations of free fatty acids, insulin and glucagon, were not different between the two groups. In conclusion, this study suggests that isoflavone-poor soy protein isolate affects cellular lipid homeostasis by the down-regulation of SREBPs and its target genes in the liver, which are involved in the synthesis of cholesterol and triglycerides.

To elucidate the mechanisms underlying the cholesterol lowering effects of PPARalpha agonists we investigated key regulators of cholesterol synthesis and uptake in rats and in the rat hepatoma cell line Fao after treatment with the PPARalpha agonists clofibrate and WY 14,643, respectively. In rat liver as well as in Fao cells, PPARalpha activation led to a decrease of transcriptionally active nuclear SREBP-2. mRNA concentrations of the key regulators of SREBP processing, Insig-1 in rat liver and Insig-1 and Insig-2a in Fao cells, were increased upon PPARalpha activation. Thus we suggest, that the observed reduction of the amount of nuclear SREBP-2 was due to an inhibition of the processing of the precursor protein. Both, in rat liver and in Fao cells, mRNA concentrations of the SREBP-2 target genes HMG-CoA reductase (EC1.1.1.34) and LDL receptor were reduced after treatment with the PPARalpha agonists. Furthermore, treatment of Fao cells with WY 14,643 reduced cholesterol synthesis. As a result, the amount of total cholesterol in liver, plasma and lipoproteins of clofibrate treated rats and in WY 14,643 treated Fao cells was decreased compared to control animals and cells, respectively. In conclusion, we could show a novel link between PPARalpha and cholesterol metabolism by demonstrating that PPARalpha activation lowers cholesterol concentration by reducing the abundance of nuclear SREBP-2.

Objective We assessed the effect of dietary proteins isolated from beef, pork, and turkey meat on concentrations of cholesterol and triacylglycerols in plasma, lipoproteins, and liver and the composition of the microsomal membrane (fatty acids, phosphatidylcholine/phosphatidylethanolamine ratio) compared with that of casein and soy protein in rats. Methods Five groups of 12 rats each were fed semisynthetic diets for 20 d that contained 200 g/kg of proteins isolated from beef, pork, or turkey meat or, as controls, casein or soy protein. Results Rats fed beef, pork, or turkey proteins did not differ in cholesterol concentrations of plasma, lipoproteins, and liver and in composition of microsomal membrane from rats fed the casein diet. All groups fed a protein from an animal source had higher very low-density lipoprotein (VLDL) and liver cholesterol concentrations than did rats fed soy protein. However, rats fed pork protein had lower concentrations of triacylglycerols in liver, plasma, and VLDL and lower mRNA concentrations of sterol regulatory element binding protein-1 and glucose-6-phosphate dehydrogenase than did rats fed casein. However, concentrations of plasma and VLDL triacylglycerols in rats fed pork protein were not as low as those observed in rats fed soy protein. Conclusion Proteins isolated from beef, pork, or turkey meat do not differ from casein in their effects on cholesterol metabolism. Pork protein decreases plasma triacylglycerol concentrations compared with casein but not compared with soy protein. The triacylglycerol-lowering effect of pork protein compared with casein is suggested to be caused by decreased hepatic fatty acid synthesis.

It has been shown that clofibrate treatment increases the carnitine concentration in the liver of rats. However, the molecular mechanism is still unknown. In this study, we observed for the first time that treatment of rats with the peroxisome proliferator activated receptor (PPAR)-alpha agonist clofibrate increases hepatic mRNA concentrations of organic cation transporters (OCTNs)-1 and -2 which act as transporters of carnitine into the cell. In rat hepatoma (Fao) cells, treatment with WY-14,643 also increased the mRNA concentration of OCTN-2. mRNA concentrations of enzymes involved in carnitine biosynthesis were not altered by treatment with the PPARalpha agonists in livers of rats and in Fao cells. We conclude that PPARalpha agonists increase carnitine concentrations in livers of rats and cells by an increased uptake of carnitine into the cell but not by an increased carnitine biosynthesis.

Conjugated linoleic acids (CLA) have attracted scientific interest due to their potential beneficial effects on atherosclerosis. Recently, a mixture of CLA isomers was demonstrated to upregulate LDL receptor expression in the human hepatoma cell line HepG2. However, the underlying mechanisms remain to be resolved. Thus, the aim of this study was to elucidate how CLA mediates upregulation of LDL receptor in HepG2 cells and whether this upregulation is isomer-specific. The results revealed that LDL receptor promoter activity and mRNA expression were strongly induced upon treatment with t10c12-CLA (P0.05), whereas c9t11-CLA and linoleic acid (LA) had no effect. In addition, only treatment with t10c12-CLA markedly induced mRNA expression of SREBP-2 and HMG-CoA reductase and slightly induced that of SREBP-1 (P0.05). Using SREBP-2 knockdown cells, we could demonstrate that the effect of t10c12-CLA on LDL receptor gene transcription was significantly reduced when compared to control cells (P0.05). When using SREBP-1 knockdown cells the effect of t10c12-CLA on LDL receptor mRNA only slightly decreased compared to control cells. In addition, using different deletion constructs of the LDL receptor gene promoter we showed that the induction of the LDL receptor by t10c12-CLA is independent of the AP-1 motif in the LDL receptor promoter. In conclusion, the present study revealed that transcriptional activation of the LDL receptor gene by t10c12-CLA is dependent on the upregulation of SREBP-2 and is probably due to the activation of the SRE-1 in the LDL receptor gene promoter in HepG2 cells. Thus, the decreased plasma cholesterol levels in response to CLA as observed in a limited number of animal and human studies might be explained by an enhanced uptake of VLDL and LDL cholesterol via hepatic LDL receptors. However, it provides no explanation for the outcome of most human studies reporting unaltered or even increased plasma and LDL cholesterol concentrations in response to supplementation with CLA.

Previous studies have shown that supplementation of sow diets with l -carnitine increases body weights of their piglets at birth. It has not yet been investigated whether piglets of sows supplemented with l -carnitine differ in their body composition or metabolic parameters from those of control sows at birth and during the suckling period. This study was performed to investigate whether supplementation of sows with l -carnitine during pregnancy and lactation influences body composition and lipid metabolism of their piglets. An experiment was conducted with 40 primiparous sows which were assigned to two groups of 20 sows each and had free access to a nutritionally adequate diet. One group was supplemented with 125 mg l -carnitine/day during pregnancy and 250 mg l -carnitine/day during lactation; the other group (control group) did not receive l -carnitine. l -Carnitine treated sows had a higher feed intake during pregnancy (P l -carnitine treated sows had fewer stillborn piglets (P l -carnitine treated sows had higher concentrations of l -carnitine in plasma and carcass at birth and on days 10 and 20 of age than control piglets (P l -carnitine improves the l -carnitine status of their piglets at birth and during the suckling period but does not influence their body composition or lipid metabolism.

Atmospheric pressure photoionisation (APPI) was used in combination with silver-ion (Ag + )-HPLC for detection of (conjugated) fatty acid methyl esters (FAME) by tandem-mass spectrometry. APPI-MS of methyl esters of conjugated linoleic acid showed an increase in signal-to-noise ratio by a factor of 40 compared to atmospheric pressure chemical ionization in the positive mode. It was possible to identify double bond position, configuration and chain length of FAME based on chromatographic separation and mass detection. The developed LC–MS method is useful for the analysis of CLA elongation and β-oxidation products, especially with trans , trans -configuration, which are difficult to analyze by conventional GC–MS techniques.

Leukocyte recruitment and adhesion to the endothelium are critical steps in the early phase of atherosclerosis. Synthetic ligands of peroxisome proliferator-activated receptors (PPARs) were shown to reduce cytokine-stimulated leukocyte–endothelial cell interactions by inhibiting the NF-κB mediated inflammatory response. Conjugated linoleic acids (CLA), which are natural ligands of PPARs, were demonstrated to have anti-inflammatory and anti-atherogenic properties in vivo. With a view to elucidating the anti-atherogenic mechanisms of CLA, the present study aimed to explore the effects of cis -9, trans -11 CLA and trans -10, cis -12 CLA on cytokine-induced chemokine release, surface expression of adhesion molecules (ICAM-1, VCAM-1, and E-selectin) and U937 monocyte adhesion in human aortic endothelial cells (HAEC). Treatment of HAECs with 2 ng/mL of TNFα markedly increased expression of adhesion molecules, U937 monocyte adhesion, and release of the monocyte chemoattractant protein (MCP)-1. However, treatment of HAECs with either CLA isomer or linoleic acid did not modulate the cytokine-induced expression of ICAM-1, VCAM-1, and E-selectin, U937 cell adhesion and MCP-1 release. In addition, both CLA isomers and linoleic acid slightly increased PPARγ DNA-binding activity, but did not alter DNA-binding activity of NF-κB. In conclusion, CLA isomers showed no effect on cytokine-induced monocyte–endothelial cell interactions and on the molecular mechanisms regulating these processes in HAEC. This study suggests that anti-atherogenic effects of CLA observed in vivo are probably not mediated by reduced monocyte–endothelial cell interactions.

Conjugated linoleic acids (CLAs) are bioactive lipid compounds showing anti-atherogenic actions in cell culture experiments and animal models of atherosclerosis without exact knowledge about the underlying mechanisms. CLAs were recently reported to be further metabolized to bioactive conjugated metabolites indicating that these metabolites are possibly involved in mediating the anti-atherogenic actions of CLA. Regarding the lack of information with respect to the formation of CLA metabolites in the vascular endothelium, which is strongly involved in the process of atherosclerosis, the present study aimed to explore the potential formation of CLA metabolites in vascular endothelial cells. The results from the present study show for the first time that the CLA isomers cis-9, trans-11 CLA and trans-10, cis-12 CLA are metabolized within endothelial cells to β-oxidation products such as CD16:2c7t9 and CD16:2t8c10 and elongation products such as CD20:2c11t13, CD20:2t12c14 as well as CD22:2c13t15 and CD22:2t14c16. Different CD16:2/CLA ratios observed between cells treated with different CLA isomers indicate that the metabolism of CLAs depends on the configuration of the conjugated double bonds. In conclusion, regarding the biological activity reported for CD20:2t12c14 and other metabolites of CLA, the present results indicate that metabolites of CLA are possibly also involved in mediating the anti-atherogenic actions of CLA.

Conjugated linoleic acids (CLAs) were reported to have anti-atherogenic properties in animal feeding experiments. In an attempt to elucidate the molecular mechanisms of these anti-atherogenic effects, the modulatory potential of CLA on cytokine-induced eicosanoid production from smooth muscle cells (SMCs), which contributes to the chronic inflammatory response associated with atherosclerosis, has been investigated in the present study. cis-9, trans-11 CLA and trans-10, cis-12 CLA were shown to reduce proportions of the eicosanoid precursor arachidonic acid in SMC total lipids and to inhibit cytokine-induced NF-kappaB DNA-binding activity, mRNA levels of inducible enzymes involved in eicosanoid formation (cPLA2, COX-2, mPGES), and the production of the prostaglandins PGE2 and PGI2 by TNFalpha-stimulated SMCs in a dose-dependent manner. The effect of 50 micromol/L of either CLA isomer was as effective as 10 micromol/L of the PPARgamma agonist troglitazone in terms of inhibiting the TNFalpha-stimulated eicosanoid production by SMCs. PPARgamma DNA-binding activity was increased by both CLA isomers compared to control cells. Moreover, it was shown that the PPARgamma antagonist T0070907 partially abrogated the inhibitory action of CLA isomers on cytokine-induced eicosanoid production and NF-kappaB DNA-binding activity by vascular SMCs suggesting that PPARgamma signalling is at least partially involved in the action of CLA in human vascular SMCs. With respect to the effects of CLA on experimental atherosclerosis, our findings suggest that the anti-inflammatory effect of CLA is at least partially responsible for the anti-atherogenic effects of CLA observed in vivo.

Conjugated linoleic acids (CLA) have attracted scientific interest due to their potential beneficial effects on atherosclerosis. Recent studies demonstrated that conjugated metabolites of CLA are found in tissues of CLA-fed animals and cultured cells treated with CLA. This observation has gained in importance since it has recently been shown that these metabolites of CLA exert specific biological activities. Therefore, the present study aimed to explore the potential formation of metabolites of cis-9, trans-11 CLA, trans-10, cis-12 CLA and trans-9, trans-11 CLA in cells of the vascular wall, which has not yet been shown. Examination of fatty acid composition of total cell lipids using Ag+-HPLC, GC-FID and GC-MS analysis revealed a significant isomer-specific formation of conjugated metabolites of CLA such as CD16:2, CD20:2 and CD22:2 in human coronary artery smooth muscle cells treated with various CLA isomers. Different CD16:2/CLA ratios between various CLA isomers as observed in the present study indicate that fatty acid metabolism is differently affected by the configuration of the double bonds. In conclusion, the observation from the present study suggests that the effects of CLA in vascular cells might not only be mediated by CLA itself but also by its conjugated metabolites. Future studies using highly purified conjugated metabolites of CLA are necessary to study their role in mediating biological effects of CLA in cell culture systems.

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; P0.05). 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 (P0.05). Various hydrolysate fractions from casein and soy protein influenced the release of 6-keto-prostaglandin F1alpha and nitric oxide (P0.05) 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.

Recent studies showed that conjugated linoleic acids (CLA) lower triacylglycerol concentrations in the milk of lactating animals. This study was performed to determine the reasons for this phenomenon; we also investigated whether there is a relation between altered lipid metabolism in the liver and the reduction in milk triacylglycerols in rats fed CLA. Two groups of female rats were fed diets containing 0 (sunflower oil (SFO) group) or 14.7 g/kg diet of a CLA mixture (CLA group) at the expense of sunflower oil during growth, pregnancy, and lactation. CLA-fed rats had 49 and 80% lower mRNA concentration and activity of fatty acid synthase, respectively, a 51% lower mRNA concentration of lipoprotein lipase (LPL) in their mammary glands at d 17 of lactation, and a 46% lower milk fat content than SFO rats (P 0.05). Although CLA rats had lower concentrations of triacylglycerols in the liver than SFO rats (20.8 2.6 vs. 62.6 27.7 mol/g, P 0.05), concentrations of triglycerides in plasma, which are the substrates of LPL, did not differ between the groups. Moreover, the number of pups per litter, litter weights, and pup weights at d 17 of lactation were 41, 35, and 22% lower, respectively, in the CLA group than in the SFO group. In conclusion, the present study suggests that dietary CLA reduces triacylglycerol concentrations in the milk via reduced de novo fatty acid synthesis in the mammary gland and an impaired uptake of fatty acids from lipoproteins into the mammary gland. This might be the reason for reduced growth rates and an increased mortality of suckling pups. J. Nutr. 134: 3327-3334, 2004.

Oxidized fats affect animal metabolism in several ways. To gain a comprehensive understanding of the molecular mechanisms underlying the effects of dietary oxidized fats in rats at varying dietary vitamin E concentrations, the gene expression profile of the liver was monitored with an array containing 1176 binding sites for cDNAs. Rats were fed diets with a fresh fat and vitamin E concentrations of 25 or 250 mg alpha-tocopherol/kg (FF25, FF250 rats) or a fat heated at 50 degrees C for 38 d, with vitamin E concentrations of 25 or 250 mg alpha-tocopherol/kg (OF25, OF250 rats) for 63 d. Differences in gene expression were considered to be significant at a ratio of at least 1.4. In the OF25 rats, the expression of 47 genes was altered; in the OF250 rats, the expression of 37 genes was altered, and in the FF250 rats, the expression of 21 genes was altered compared with FF25 rats. In both OF25 and OF250 rats, a series of target genes of the peroxisome proliferator-activated receptor alpha (PPAR alpha) was upregulated. Determination of gene expression of acyl CoA oxidase and activity of catalase confirmed that oxidized fats caused peroxisome proliferation in the liver. In OF25 and OF250 rats, there was also upregulation of 12 and 5 genes involved in xenobiotic metabolism and stress response, of 7 and 7 genes involved in protein metabolism, of 5 and 2 genes encoding intracellular effectors or modulators and of 5 and 6 genes, respectively, encoding activators or repressors of transcription or translation. In conclusion, this study provides indirect evidence that dietary oxidized fats cause an activation of the PPAR alpha, irrespective of the dietary vitamin E concentration. Identification of several other differentially regulated genes may be helpful to understand the effects of oxidized fats on animal metabolism.

The present study was undertaken to find out whether the tryptophan requirement of laying hens is influenced by the supply of large neutral amino acids (LNAA). A factorial experiment was performed in which the dietary tryptophan concentration was varied at six different levels (1.0, 1.25, 1.5, 1.75, 2.0, and 2.5 g tryptophan/kg diet). As the second factor, the dietary concentrations of LNAA (isoleucine, valine, leucine, phenylalanine, and tyrosine) were varied at two levels. The first level provided an adequate supply of these amino acids; at the second level the concentrations of these amino acids were 40% higher than at the first level. The tryptophan requirement was estimated by a broken-line model and an exponential model of regression analysis. The tryptophan intake required for optimum (100% of maximum in the broken-line model, 95% of the maximum in the exponential model) egg production and daily egg mass was lower in hens fed the diets with high LNAA concentrations (145 and 155 mg/hen per day, respectively, in average of both models) than in hens fed the diets with adequate concentrations of LNAA (184 and 198 mg/hen per day, respectively, in average of both models). In contrast, the tryptophan requirement for optimum BW gain was lower in hens fed the diets with adequate LNAA concentrations (178 mg tryptophan per day) than in hens fed the diets with a high concentration of LNAA (212 mg tryptophan per day). In conclusion, the study suggests that an interaction between dietary LNAA and tryptophan exists in laying hens.

The present study was undertaken to investigate the interactions among the supplies of isoleucine, leucine, valine, and tryptophan in laying hens. A three-factor trial was conducted with laying hens in which the dietary concentrations of isoleucine (5.7, 8.0, and 11.5 g/kg), valine and leucine (6.3 and 7.2 g/kg and 10.1 and 11.5 g/kg, respectively), and tryptophan (1.5 and 2.4 g/kg) were varied. At the lowest concentration of valine + leucine, an increase in dietary isoleucine concentration led to a dose-dependent reduction in feed consumption, daily egg mass, and body weight gain and an increase of the isoleucine concentration in plasma. At a high dietary concentration of valine + leucine, excess dietary isoleucine concentration caused only a weak depression of performance parameters; the isoleucine concentration in plasma was independent of the dietary isoleucine concentration. Increasing the dietary tryptophan concentration did not influence the effect of an excessive dietary isoleucine concentration on performance parameters. Increasing the tryptophan concentration from 1.5 to 2.4 g/kg diet did, however, lead to a significant increase in feed consumption, irrespective of the supply of isoleucine, valine, and leucine. In conclusion, our study demonstrates that the supply of valine + leucine influenced the effects of excess dietary isoleucine in laying hens, whereas the supply with tryptophan did not.

Three production trials and one nitrogen balance trial were conducted with Lohmann Brown hens to determine the requirement for and effects of an excess of isoleucine in layers at different ages (24 to 32 and 46 to 54 wk of age). The trials were designed as dose-response studies where isoleucine-deficient basal rations with 11.4 MJ metabolizable energy per kilogram were supplemented with varying amounts of L-isoleucine. In the production trials, dietary isoleucine concentrations ranged from 0.37 to 1.05%. In the three production trials, maximum daily egg mass was achieved at dietary isoleucine concentrations of between 0.39 and 0.75% (25 to 32 wk of age, daily egg mass 53 g), 0.40 and 0.57% (24 to 32 wk of age, daily egg mass 57 g), and 0.40 and 0.81% (46 to 54 wk of age, daily egg mass 56 g). The corresponding ranges of daily isoleucine intakes were 412 to 770 mg, 436 to 624 mg, and 431 to 874 mg. In the nitrogen balance trial, maximum total nitrogen retention was achieved at dietary isoleucine concentrations of between 0.43 and 0.57%. Dietary isoleucine concentrations higher than 0.8% caused a reduction in hen BW. Dietary isoleucine concentrations higher than 1.0% additionally caused a reduction in the daily egg mass. The study thus shows that the margin between requirement and excess of isoleucine is narrow in laying hens.

In vitro studies suggest that high vitamin E supplementation has prooxidative activity, but very few studies have investigated this effect in vivo. We investigated the effect of excess vitamin E on the antioxidative status of rat erythrocytes and indicators of hemolysis. Six groups of growing male Sprague-Dawley rats were fed purified diets with three different vitamin E doses [100, 1000 and 10,000 mg all-rac-alpha-tocopheryl acetate (TA)/kg diet] and two different dietary fats (salmon oil and lard) for 8 wk. The rats whose diet contained salmon oil and 10,000 mg TA/kg had lower activities of superoxide dismutase (P < 0.05), glutathione peroxidase (P < 0.05), catalase (P < 0.05) and glucose-6-phosphate dehydrogenase (P < 0.05) and a lower concentration of glutathione (P < 0.05) in the erythrocyte cytosol than rats whose diet contained 100 mg TA/kg. The concentration of free hemoglobin and the binding capacity of haptoglobin in plasma, both indicators of in vivo hemolysis, did not differ between rats fed the salmon oil diet with 100 or 10,000 mg TA/kg. In the rats whose diet contained lard, the activities of antioxidant enzymes in erythrocytes and indicators of in vivo hemolysis were independent of the dietary vitamin E concentration. The results of the study suggest that an excessive vitamin E intake, when combined with salmon oil in the diet, lowers the activities of antioxidant enzymes in erythrocytes without affecting in vivo hemolysis.

The objective of this study was to determine the effects of cis-9,trans-11 and trans-10,cis-12 CLA on the fatty acid desaturation in a human hepatoma cell line, HepG2. Therefore, experiments were conducted in which HepG2 cells were incubated with various concentrations of those fatty acids and the concentrations of fatty acids in various lipid fractions of HepG2 cells were determined. In the presence of linoleic acid as substrate, cells treated with 25 micromol/L of trans-10,cis-12 CLA had lower ratios of dihomo-gamma-linoleic acid to linoleic acid and of arachidonic acid to linoleic acid in phospholipids than control cells; with alpha-linolenic acid as substrate, they had a lower ratio of eicosapentaenoic acid to alpha-linolenic acid in phospholipids than control cells. Cells treated with cis-9,trans-11 CLA did not differ in these ratios from control cells. Cells treated with trans-10,cis-12 CLA had also a markedly lower ratio of monounsaturated fatty acids (MUFA) to saturated fatty acids (SFA) in lipids than control cells; cells treated with cis-9,trans-11 CLA had a slightly lower MUFA:SFA ratio than control cells. These findings suggest that trans-10,cis-12 CLA suppresses Delta9-, Delta6- and Delta5-desaturation in HepG2 cells; cis-9,trans-11 CLA slightly reduces Delta9-desaturation but does not inhibit Delta6- and Delta5-desaturation. Moreover, HepG2 cells treated with 100 micromol/L of trans-10,cis-12 CLA released larger amounts of 6-keto-prostaglandin F(1alpha) and prostaglandin F(2alpha) than control cells. Treatment of cells with cis-9,trans-11 CLA did not alter the release of these eicosanoids compared with control cells. In conclusion, this study suggests that trans-10,cis-12 CLA has significant effects on the metabolism of essential fatty acids in HepG2 cells, whereas cis-9, trans-11 CLA does not have any effect in this respect.

A recent study demonstrated that feeding a diet with a thermally oxidized fat increases the concentration of thyroxine in plasma of miniature pigs. This study was undertaken to investigate whether the effect of thermally oxidized fats on plasma thyroid hormones is influenced by the supply of vitamin E or selenium. Two experiments were conducted using male Sprague-Dawley rats. The first experiment included eight groups of rats fed diets with either fresh fat or three different types of oxidized fat prepared by heating at 50 degrees C, 105 degrees C or 190 degrees C for 42 d. The diets contained either 25 or 250 mg alpha-tocopherol equivalents per kg. The second experiment included four groups of rats fed diets with fresh fat or oxidized fat heated at 55 degrees C, containing either 70 or 570 microg selenium per kg for 56 d. Rats fed all types of oxidized fats had higher concentrations of free and total thyroxine in plasma than rats fed the equivalent diets with fresh oil; the concentrations of triiodothyronine and thyroid-stimulating hormone did not differ between rats fed fresh and those fed oxidized fats. The effect of the oxidized fat on the plasma thyroxine concentration was completely independent of the supply of vitamin E (expt. 1) and the supply of selenium (expt. 2). Our results confirm that oxidized dietary fats raise the plasma thyroxine concentration and show that this phenomenon is independent of the vitamin E and selenium status.

article i nfo a bstract Two experiments were conducted to examine the effect of benzoic acid in the diet of lactating sows on urinary pH and nutrient digestibility. In the first experiment, three levels of benzoic acid in the diet were tested (0.5, 1.0, and 2.0%), while in the second experiment only one dietary level of benzoic acid (0.5%) was used. In the second experiment, the urinary excretion of benzoic acid and hippuric acid was additionally measured. The digestibility was estimated in experiment 1 with HCl-insoluble ash as a marker and in experiment 2 in a total balance period with collection of urine and faeces over 5 days. Supplementation of benzoic acid reduced the urinary pH in a dose dependent manner (pb0.01). The relationship between benzoic acid intake and urinary pH response was linear. Dietary benzoic acid at a level of 2% increased the digestibility of organic matter, protein, fat and fiber in the diet. In sows fed the diet supplemented with 0.5% of benzoic acid, 64% of benzoic acid ingested was excreted via urine as hippuric acid, the remainder as unmetabolized benzoic acid.

The present study was performed to investigate the effect of zinc deficiency on concentrations of lipids in plasma and lipoproteins and the susceptibility of low-density lipoproteins (LDL) to lipid peroxidation in rats fed diets containing either linseed oil or olive oil as dietary fat, using a bifactorial experimental design. To ensure an adequate food intake, all the rats were force-fed by gastric tube. The dietary fat had a stronger effect on those parameters than the zinc supply. Rats fed linseed oil had lower concentrations of cholesterol, triglycerides, and phospholipids in plasma and lipoproteins but a higher susceptibility of LDL to lipid peroxidation than rats fed olive oil. The effect of zinc deficiency on those parameters were modified by the dietary fat. Zinc-deficient rats fed linseed oil had increased concentrations of total lipids, cholesterol, and phospholipids in plasma as well as in low- and high-density lipoproteins and an increased susceptibility of LDL to copper-induced lipid peroxidation compared to zinc-adequate rats fed linseed oil. The increased susceptibility of LDL in those rats might be attributable to increased levels of arachidonic acid and eicosapentaenoic acid in LDL. In contrast, in zinc-deficient rats fed olive oil, concentrations of lipids in plasma and lipoproteins were only slightly changed and the susceptibility of LDL to copper-induced lipid peroxidation was reduced compared to zinc-adequate rats fed olive oil. Tocopherol concentrations in plasma, expressed per mol lipid, were not influenced by zinc deficiency in the rats fed both types of fat. Therefore, a changed vitamin E status might not be involved in the effects of zinc deficiency on the susceptibility of LDL. In conclusion the study shows that the effects of zinc deficiency on the concentrations of plasma lipids and the susceptibility of LDL to lipid peroxidation depend on the type of fat. Therefore, the type of dietary fat must be given attention in the investigation of the effects of zinc deficiency on parameters of lipid metabolism.

Several clinical features of zinc deficiency in rats are similar to those of essential fatty acid deficiency suggesting a role of zinc in fatty acid metabolism. In order to find out the role of zinc in fatty acid metabolism, several zinc deficiency studies in rats have been carried out. The results of those studies are reviewed in this paper with respect to the effects of zinc deficiency on the desaturation of fatty acids, the incorporation of polyunsaturated fatty acids into tissue lipids and the fatty acid composition of erythrocyte membranes. Zinc deficiency experiments indicate that zinc plays a role in Δ5 and Δ6 desaturation although there are some reports that did not support such a function of zinc. The effects of zinc deficiency on Δ9 desaturation are contradictory. Some results suggested that zinc deficiency increased Δ9 desaturation, while others suggested reduced Δ9 desaturation. Phospholipids of zinc deficient rats contained higher amounts of (n-3) polyunsaturated fatty acids and lower amounts of (n-6) polyunsaturated fatty acids, suggesting that zinc deficiency influences the incorporation of polyunsaturated fatty acids into phospholipids. In contrast, zinc deficiency had only slight effects on the fatty acid composition of erythrocyte membranes. Consequences of changes in fatty acid metabolism by zinc deficiency may include synthesis of eicosanoids and properties of membranes.

The aim of the present study was to investigate if zinc-deficiency influences the regulation of lipogenic enzymes by dietary polyunsaturated fatty acids. Therefore, rats were fed a fat-free diet with either adequate or deficient zinc supply for 6 days. After that period the groups were divided; half of the rats were given the fat-free zinc-adequate and zinc-deficient diets for another 3 days, whereas the other half was given the same diets supplemented with 5% safflower oil. To control food intake, all the rats were force-fed by gastric tube. At the end of the experiment, zinc-deficient rats fed both, the fat-free diet and the 5% safflower oil diet had largely reduced zinc concentrations and activities of alkaline phosphatase in serum proving their zinc-deficient state. Zinc-deficient rats fed both the fat-free and the 5% safflower oil diet had markedly increased concentrations of triglycerides in liver compared with zinc-adequate rats. Zinc-deficient rats fed both the fat-free diet and the 5% safflower oil diet had increased activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase compared with their controls. In contrast, activities of fatty acid synthase and malic enzyme were not changed by zinc deficiency. This suggests that fatty liver is not mainly due to increased lipogenesis but to other factors such as impaired exclusion of lipids from liver. The addition of safflower oil to the fat-free diet suppressed activities of hepatic lipogenic enzymes in both, zinc-adequate and zinc-deficient rats. However, the suppression was more pronounced in zinc-adequate rats than in zinc-deficient rats. In adipose tissue, addition of safflower oil elevated activities of lipogenic enzymes in zinc-adequate rats but lowered activities in zinc-deficient rats. Those data suggest that zinc-deficiency affects regulation of lipogenic enzymes in liver and adipose tissue.

The full process of fatty acid methyl ester (FAME) analysis consists of esterification of lipids, and of injection, separation, identification and quantitation of the FAMEs. In order for the required accuracy and precision to be attained, each of these steps has to be optimized. Esterification of lipids can be carried out with several reagents based on acid-catalysed or base-catalysed reactions. The advantages and disadvantages of these reagents are discussed. The most critical step in the gas chromatographic analysis of FAMEs is sample introduction. The classical split injection technique, which is the most widely used technique in the analysis of FAMEs, has the potential disadvantage of boiling-point-dependent sample discrimination. Cold injection of the sample, either on-column or by programmed-temperature vaporization, does not present this problem and should therefore be preferred. Modern, commercially available fused-silica capillary columns offer excellent separation of FAMEs from biological samples. Very polar stationary phases give excellent separation of all FAMEs but have relatively low thermal stability, resulting in long retention times. Non-polar phases have a much greater thermal stability but inferior selectivity. For many analyses, phases of intermediate polarity, which combine the advantages of a relatively high resolution capability with relatively high thermal stability, are the most suitable. FAMEs can be identified by comparison of their retention times with those of individual purified standards or secondary standards based on lipids that have been well characterized in literature. Relative retention times and equivalent chain-length values also provide useful information. FAMEs can be quantitated by peak areas via calibration factors, and absolute concentrations can be determined by adding an internal standard. Among numerous applications in biomedical research, the analysis of fatty acids from body tissues may contribute to the understanding of the link between the dietary intake of fatty acids and the diseases with which these acids are associated.