Jan Stagsted

A short-term intervention trial with selenate, selenium-enriched yeast and selenium-enriched milk: effects on oxidative defence regulation.

Increased Se intakes have been associated with decreased risk of cancer and CVD. Several mechanisms have been proposed, including antioxidant effects through selenoproteins, induction of carcinogen metabolism and effects on the blood lipid profile. In a 4 x 1 week randomised, double-blind cross-over study, healthy young men supplemented their usual diet with selenate, Se-enriched yeast, Se-enriched milk or placebo (Se dose was 300 microg/d for selenate and Se-enriched yeast, and about 480 microg/d for Se-enriched milk) followed by 8-week washout periods. All Se sources increased serum Se levels after supplementation for 1 week. The effect of the organic forms did not differ significantly and both increased serum Se more than selenate. Conversely, thrombocyte glutathione peroxidase (GPX) was increased in the periods where subjects were supplemented with selenate but not in those where they were given Se-enriched yeast or Se-enriched milk. We found no effect on plasma lipid resistance to oxidation, total cholesterol, TAG, HDL- and LDL-cholesterol, GPX, glutathione reductase (GR) and glutathione S-transferase (GST) activities measured in erythrocytes, GPX and GR activities determined in plasma, or GR and GST activities in thrombocytes. Leucocyte expression of genes encoding selenoproteins (GPX1, TrR1 and SelP), and of electrophile response element-regulated genes (GCLC, Fra1 and NQO1) were likewise unaffected at all time points following intervention. We conclude that thrombocyte GPX is specifically increased by short-term selenate supplementation, but not by short-term supplementation with organic Se. Short-term Se supplementation does not seem to affect blood lipid markers or expression and activity of selected enzymes and a transcription factor involved in glutathione-mediated detoxification and antioxidation.

Changes in the gut microbiota of cloned and non-cloned control pigs during development of obesity: gut microbiota during development of obesity in cloned pigs

BackgroundObesity induced by a high-caloric diet has previously been associated with changes in the gut microbiota in mice and in humans. In this study, pigs were cloned to minimize genetic and biological variation among the animals with the aim of developing a controlled metabolomic model suitable for a diet-intervention study. Cloning of pigs may be an attractive way to reduce genetic influences when investigating the effect of diet and obesity on different physiological sites. The aim of this study was to assess and compare the changes in the composition of the gut microbiota of cloned vs. non-cloned pigs during development of obesity by a high-fat/high-caloric diet. Furthermore, we investigated the association between diet-induced obesity and the relative abundance of the phyla Firmicutes and Bacteroidetes in the fecal-microbiota. The fecal microbiota from obese cloned (n = 5) and non-cloned control pigs (n= 6) was investigated biweekly over a period of 136 days, by terminal restriction fragment length polymorphism (T-RFLP) and quantitative real time PCR (qPCR).ResultsA positive correlation was observed between body-weight at endpoint and percent body-fat in cloned (r=0.9, P<0.0001) and in non-cloned control pigs (r=0.9, P<0.0001). Shannon Weaver and principal component analysis (PCA) of the terminal restriction fragments (T-RFs) revealed no differences in the bacterial composition or variability of the fecal microbiota between the cloned pigs or between cloned and non-cloned control pigs. Body-weight correlated positively with the relative abundance of Firmicutes in both cloned (r=0.37; P<0.02) and non cloned-control pigs (r=0.45; P<0.006), and negatively with the abundance of Bacteroidetes in cloned pigs (r=−0.33, P<0.04), but not in the non-cloned control pigs.ConclusionThe cloned pigs did not have reduced inter-individual variation as compared to non-cloned pigs in regard to their gut microbiota in neither the obese nor the lean state. Diet-induced obesity was associated with an increase in the relative abundance of Firmicutes over time. Our results suggest that cloned pigs are not a more suitable animal model for gut microbiota-obesity related studies than non-cloned pigs. This study is the first to evaluate if cloned pigs provide a better animal model than conventional pigs in diet-intervention, obesity and gut microbiota research.

Colostrum and bioactive, colostral peptides differentially modulate the innate immune response of intestinal epithelial cells.

Characterization and identification of peptides with bioactivity from food have received considerable interest recently since such bioactive components must be adequately documented if they are part of functional food claims. We have characterized peptides from colostrum or those generated by a simulated gastrointestinal digest (GI) and tested them for bioactivity using murine intestinal (mICc12) cells and compared with bioactivity of intact colostrum. The peptides were recovered in the permeate after dialysis. The presence of peptides in the permeate was confirmed by C18 RP‐HPLC, determination of free amino termini and MALDI MS. The bioactivity of the intact colostrum and colostral peptides in the permeate was tested using mICc12 cells stimulated in the absence or presence of different bacterial ligands that mediate cellular activation through stimulation of Toll‐like receptors (TLR). Whereas intact colostrum generally reduced TLR‐mediated signaling, the isolated peptides seemed to either stimulate or reduce the immune response depending on the bacterial ligand used for stimulation. Interestingly, the most potent bioactive peptides originated from nondigested colostrum, which had only been subject to endogenous protease activity. Identified peptides in the nondigested colostrum originated exclusively from the casein fraction of colostrum as shown by MALDI MS/MS identification. Thus, multiple components with different bioactivities towards the innate immune response appear in bovine colostrum. Copyright

Characterization of Peroxides Formed by Riboflavin and Light Exposure of Milk. Detection of Urate Hydroperoxide as a Novel Oxidation Product

Characterization of peroxides by size exclusion chromatography (SEC) of milk following exposure to riboflavin and light showed that hydrogen peroxide was the most abundant peroxide formed since it could be removed by catalase. Formation of peroxides after separation by SEC showed that hydrogen peroxide formation was primarily increased in the presence of caseins and ascorbate, although whey proteins also were found to contribute. Caseins and beta-lactoglobulin also formed catalase-resistant peroxides, presumably protein hydroperoxides. A catalase-resistant and unstable peroxide was observed in fractions containing urate. Experiments performed with pure urate suggested that urate radicals reacted further with superoxide leading to a urate hydroperoxide. Electron paramagnetic resonance spectroscopy using spin-traps showed that the presence of oxygen was required for urate radical formation, which could be assigned as nitrogen-centered radicals. These results suggest a new route during light-induced oxidation sensitized by flavins, in effect making urate pro-oxidative.

Orosomucoid expression profiles in liver, adipose tissues and serum of lean and obese domestic pigs, Göttingen minipigs and Ossabaw minipigs

The acute phase protein orosomucoid (ORM) has anti-inflammatory and immunomodulatory effects, and may play an important role in the maintenance of metabolic homeostasis in obesity-induced low-grade inflammation. Even though the pig is a widely used model for obesity related metabolic symptoms, the expression of ORM has not yet been characterized in such pig models. The objective of this study was to investigate the expression of ORM1 mRNA in liver, visceral adipose tissue, subcutaneous adipose tissue (SAT) from the abdomen or retroperitoneal abdominal adipose tissue (RPAT) and SAT from the neck, as well as the serum concentration of ORM protein in three porcine obesity models; the domestic pig, Göttingen minipigs and Ossabaw minipigs. No changes in ORM1 mRNA expression were observed in obese pigs compared to lean pigs in the four types of tissues. However, obese Ossabaw minipigs, but none of the other breeds, showed significantly elevated ORM serum concentrations compared to their lean counterparts. Studies in humans have shown that the expression of ORM was unchanged in adipose tissue depots in obese humans with an increased serum concentration of ORM. Thus in this respect, obese Ossabaw minipigs behave more similarly to obese humans than the other two pig breeds investigated.

Journey beyond immunology. Regulation of receptor internalization by major histocompatibility complex class I (MHC‐I) and effect of peptides derived from MHC‐I

Certainly, the MHC-I molecule has an indisputable immunological role in binding and presenting peptides to cytotoxicT cells. This is comprehensively documented and wellunderstood at the molecular level (28). Some of these aspects are treated in later sections. The thesis’ hypothesis, however, implicates a basic regulatory role of MHC-I that takes the function of this molecule beyond immunology. Many attempts have been made to demonstrate a more general role of MHC-I, but so far none have succeeded indisputably (see also our papers (2,6) for hrther discussion). Our use of MHC-I derived peptides represents an attempt to address this issue in a novel way. I started out on this journey beyond immunology in 1987 at University of California, Berkeley. This was the time in which MHC restriction moved into the realm of molecular understanding (29-31). The feasibility of binding peptides directly to MHC stimulated immunologists to use small peptides derived from different parts of MHC-I to probe MHC-I restriction and allorecognition by cytotoxic T-lymphocytes, i.e. the requirement for same MHC-I haplotype of effector and target cells to evoke lymphocyte-mediated killing of virally infected cells. We also knew that the structure of an MHC-I molecule had been solved by Don Wiley’s group. It was evident that breakthroughs in understanding central issues in immunology were coming, and we were all excited about these developments. Maybe peptides derived from MHC-I could mimic or possibly compete other MHC-I interactions, apart from the reaction with the T-cell receptor that fascinates immunologists. We did some long-shot experiments, injected the MHC-I peptides intravenously into mice and monitored their blood glucose levels. I obtained incredible effects (figure l), with some mice going into convulsions due to ex-tremely low blood glucosevalues, just as those obtained with an overdose of insulin (insulin-chock). Such results naturally off-set large scale studies, which eventually formed the basis for our Cell paper (2), and with these preliminary results in hand, we started probing for mechanism of action. How these extraordinary effects were obtained became of utmost importance. As blood glucose levels are normally regulated by insulin in the intact animal, we focused for a while on the signaling activity of the insulin receptor (1). Before discussing this work, I will briefly review the insulin receptor, which was central to our work on elucidating the mechanism of action of the MHC-I peptides.

Liquid chromatography-mass spectrometry based metabolomics study of cloned versus normal pigs fed either restricted or ad libitum high-energy diets.

Genetically identical cloned pigs should in principle eliminate biological variation and provide more pronounced effects when subjected to, e.g., dietary interventions, but little is known about how phenotype and phenotypic variation is affected by cloning. Therefore, an investigation of the metabolome of cloned pigs compared to normal control pigs was performed to elucidate the variation and possible differences in the metabolic phenotypes during a dietary intervention. A total of 19 control pigs and 17 cloned pigs were given the same high-energy dense diet either ad libitum or in a restricted manner (60% of ad libitum) for ∼6 months, and plasma was subjected to liquid chromatography-mass spectrometry nontargeted metabolomics and biochemical analyses. Low systemic levels of IGF-1 could indicate altered growth conditions and energy metabolism in cloned pigs. In response to ad libitum feeding, clones had a decreased energy intake and lower weight gain compared to controls, and plasma lipid profiles were changed accordingly. Elevated lactate and decreased creatine levels implied an increased anaerobic metabolism in ad libitum fed clones. Less interindividual variation between cloned pigs was however not established, suggesting a strong role for epigenetics and/or the gut microbiota to develop variation.

The effect of high-fat diet on the composition of the gut microbiota in cloned and non-cloned pigs of lean and obese phenotype.

The aim of this study was to investigate the effect of high-far-high-energy diet on cloned and non-cloned domestic pigs of both lean and obese phenotype and to evaluate if the lean cloned pigs had a lower inter-individual variation as compared with non-cloned pigs. The microbiota of colon and terminal ileum was investigated in cloned and non-cloned pigs that received a high-far-high-energy diet with either restricted or ad libitum access to feed, resulting in lean and obese phenotypes, respectively. The fecal microbiota of lean pigs was investigated by terminal restriction fragment length polymorphism (T-RFLP). The intestinal microbiota of lean and obese cloned and non-cloned pigs was analyzed by quantitative real time PCR and a novel high-throughput qPCR platform (Fluidigm). Principal component analysis (PCA) of the T-RFLP profiles revealed that lean cloned and non-cloned pigs had a different overall composition of their gut microbiota. The colon of lean cloned pigs contained relatively more bacteria belonging to the phylum Firmicutes and less from the phylum Bacteroidetes than obese cloned pigs as estimated by qPCR. Fluidigm qPCR results revealed differences in specific bacterial groups in the gut microbiota of both lean and obese pigs. Our results suggest that high-far-high-energy diet is associated with changes in the gut microbiota even in the absence of obesity. Overall, the cloned pigs had a different gut microbiota from that of non-cloned pigs. To our knowledge this is the first study to investigate the gut microbiota of cloned domestic pigs of lean and obese phenotype.

Metabolomic phenotyping of a cloned pig model

BackgroundPigs are widely used as models for human physiological changes in intervention studies, because of the close resemblance between human and porcine physiology and the high degree of experimental control when using an animal model. Cloned animals have, in principle, identical genotypes and possibly also phenotypes and this offer an extra level of experimental control which could possibly make them a desirable tool for intervention studies. Therefore, in the present study, we address how phenotype and phenotypic variation is affected by cloning, through comparison of cloned pigs and normal outbred pigs.ResultsThe metabolic phenotype of cloned pigs (n = 5) was for the first time elucidated by nuclear magnetic resonance (NMR)-based metabolomic analysis of multiple bio-fluids including plasma, bile and urine. The metabolic phenotype of the cloned pigs was compared with normal outbred pigs (n = 6) by multivariate data analysis, which revealed differences in the metabolic phenotypes. Plasma lactate was higher for cloned vs control pigs, while multiple metabolites were altered in the bile. However a lower inter-individual variability for cloned pigs compared with control pigs could not be established.ConclusionsFrom the present study we conclude that cloned and normal outbred pigs are phenotypically different. However, it cannot be concluded that the use of cloned animals will reduce the inter-individual variation in intervention studies, though this is based on a limited number of animals.

Short-term effects of selenium supplementation of cows' feed on the content and distribution of selenium, copper and zinc in bovine milk, whey and blood plasma.

The effect of selenium supplementation of feed on the Se content in bovine milk, whey and plasma, and on the distribution of Se, Zn and Cu in whey and plasma was investigated. In a cross-over study two groups of cows were given a basal feed with 0.16 ppm selenite (approx. 3 mg Se/d) with or without 25 mg yeast Se/d for 2 weeks. In the supplemented group the Se content increased 10-fold in milk, 10-fold in whey and 2-fold in plasma, and after the cessation of the supplementation, selenium in milk decreased with a calculated half-life of 3.5 d. In another experiment, two groups of cows were given either 100 mg yeast Se/d for 1 week or only the basal feed. The increase in Se content in both whole and defatted milk was 40-50-fold, and in whey it was approx. 20-fold. Size-exclusion chromatography of whey using inductively coupled plasma mass spectrometry for detection showed that supplementation increased the proportion of Se in the beta-lactoglobulin-alpha-lactalbumin fraction. Distribution of Cu and Zn was essentially unaffected. In plasma, supplementation increased the Se content in all major Se fractions like selenoprotein P, albumin and low-molecular-weight compounds, but the distribution profiles of Zn and Cu underwent no major changes. The study showed for the first time the rapid kinetics of the Se increase and decrease in milk after the initiation and cessation of supplementation, respectively, and the preferential appearance of Se in the beta-lactoglobulin-alpha-lactalbumin fraction of whey. Milk highly enriched in selenium will be a useful tool for different research purposes.