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EgeUn###, beta-Carotene intake and tissue/blood concentrations have been associated with reduced incidence of several chronic diseases. Further bioactive carotenoid-metabolites can modulate the expression of specific genes mainly via the nuclear hormone receptors: retinoic acid receptor- and retinoid X receptor-mediated signalling. To better understand the metabolic conversion of beta-carotene, inter-individual differences regarding beta-carotene bioavailability and bioactivity are key steps that determine its further metabolism and bioactivation and mediated signalling. Major carotenoid metabolites, the retinoids, can be stored as esters or further oxidised and excreted via phase 2 metabolism pathways. In this review, we aim to highlight the major critical control points that determine the fate of beta-carotene in the human body, with a special emphasis on beta-carotene oxygenase 1. The hypothesis that higher dietary beta-carotene intake and serum level results in higher beta-carotene-mediated signalling is partly questioned. Alternative autoregulatory mechanisms in beta-carotene / retinoid-mediated signalling are highlighted to better predict and optimise nutritional strategies involving beta-carotene-related health beneficial mediated effects., Nutr Soc, Scottish Sect, EU-COST action POSITIVE [FA 1403]; EU-COST action EUROCAROTEN [CA 15136], The insights from EU-COST action POSITIVE (FA 1403) and EUROCAROTEN (CA 15136) are much appreciated.

There is a general agreement that granulosa cell apoptosis is the cause of antral follicle attrition. Less clear is whether this pathway is also activated in case of preantral follicle degeneration, as several reports mention that the incidence of granulosa cell apoptosis in preantral follicles is negligible. Our objective is therefore to determine which cell-death pathways are involved in preantral and antral follicular degeneration.Atretic preantal and antral follicles were investigated using immunohistochemistry and laser-capture microdissection followed by quantitative real-time reverse transcription polymerase chain reaction. Microtubule-associated light-chain protein 3 (LC3), sequestosome 1 (SQSTM1/P62), Beclin1, autophagy-related protein 7 (ATG7), and cleaved caspase 3 (cCASP3) were used as markers for autophagy and apoptosis, respectively. P62 immunostaining was far less intense in granulosa cells of atretic compared to healthy preantral follicles, while no difference in LC3 and BECLIN1 immunostaining intensity was observed. This difference in P62 immunostaining was not observed in atretic antral follicles. mRNA levels of LC3 and P62 were not different between healthy and atretic (pre)antral follicles. ATG7 immunostaining was observed in granulosa cells of preantral atretic follicles, not in granulosa cells of degenerating antral follicles. The number of cCASP3-positive cells was negligible in preantral atretic follicles, while numerous in atretic antral follicles. Taken together, we conclude that preantral and antral follicular atresia is the result of activation of different cell-death pathways as antral follicular degeneration is initiated by massive granulosa cell apoptosis, while preantral follicular atresia occurs mainly via enhanced granulosa cell autophagy.

Starches of low and high digestibility have different metabolic effects. Here, we examined whether this gives differential metabolic programming when fed in the immediate post-weaning period. Chow-fed mice were time-mated, and their nests were standardized and cross-fostered at postnatal days 1&ndash, 2. After postnatal week (PW) 3, individually housed female and male offspring were switched to a lowly-digestible (LDD) or highly-digestible starch diet (HDD) for three weeks. All of the mice received the same high-fat diet (HFD) for nine weeks thereafter. Energy and substrate metabolism and carbohydrate fermentation were studied at the end of the HDD/LDD and HFD periods by extended indirect calorimetry. Glucose tolerance (PW 11) and metabolic flexibility (PW14) were analyzed. Directly in response to the LDD versus the HDD, females showed smaller adipocytes with less crown-like structures in gonadal white adipose tissue, while males had a lower fat mass and higher whole body fat oxidation levels. Both LDD-fed females and males showed an enlarged intestinal tract. Although most of the phenotypical differences disappeared in adulthood in both sexes, females exposed to LDD versus HDD in the early post-weaning period showed improved metabolic flexibility in adulthood. Cumulatively, these results suggest that the type of starch introduced after weaning could, at least in females, program later-life health.

Colostrum is the first ingested sole nutritional source for the newborn infant. The vitamin profile of colostrum depends on the maternal vitamin status, which in turn is influenced by diet and lifestyle. Yet, the relationship between maternal vitamin status and colostrum vitamin composition has not been systematically reviewed. This review was conducted with the aim to generate a comprehensive overview on the relationship between maternal serum (plasma) vitamin concentration and corresponding colostrum composition. Three electronic databases, Embase (Ovid), Medline (Ovid), and Cochrane, were systematically searched based on predefined inclusion and exclusion criteria. Finally, a total of 11 eligible publications were included that examined the vitamins A, C, D, E, and K in both biological fluids. Maternal vitamin A, D, E, and K blood levels were unrelated to colostrum content of the respective vitamins, and serum vitamin A was inversely correlated with colostrum vitamin E. Colostrum versus maternal serum vitamins were higher for vitamins A, C, and K, lower for vitamin D, and divergent results were reported for vitamin E levels. Colostrum appears typically enriched in vitamin A, C, and K compared to maternal serum, possibly indicative of active mammary gland transport mechanisms. Inter-individual and inter-study high variability in colostrum’s vitamin content endorses its sensitivity to external factors.

Carnitine/choline acyltransferases play diverse roles in energy metabolism and neuronal signalling. Our knowledge of their evolutionary relationships, important for functional understanding, is incomplete. Therefore, we aimed to determine the evolutionary relationships of these eukaryotic transferases. We performed extensive phylogenetic and intron position analyses. We found that mammalian intramitochondrial CPT2 is most closely related to cytosolic yeast carnitine transferases (Sc-YAT1 and 2), whereas the other members of the family are related to intraorganellar yeast Sc-CAT2. Therefore, the cytosolically active CPT1 more closely resembles intramitochondrial ancestors than CPT2. The choline acetyltransferase is closely related to carnitine acetyltransferase and shows lower evolutionary rates than long chain acyltransferases. In the CPT1 family several duplications occurred during animal radiation, leading to the isoforms CPT1A, CPT1B and CPT1C. In addition, we found five CPT1-like genes in Caenorhabditis elegans that strongly group to the CPT1 family. The long branch leading to mammalian brain isoform CPT1C suggests that either strong positive or relaxed evolution has taken place on this node. The presented evolutionary delineation of carnitine/choline acyltransferases adds to current knowledge on their functions and provides tangible leads for further experimental research.

Challenge tests stress homeostasis and may reveal deviations in health that remain masked under unchallenged conditions. Ideally, challenge tests are non-invasive and applicable in an early phase of an animal experiment. Oxygen restriction (OxR; based on ambient, mild normobaric hypoxia) is a non-invasive challenge test that measures the flexibility to adapt metabolism. Metabolic inflexibility is one of the hallmarks of the metabolic syndrome. To test whether OxR can be used to reveal early diet-induced health effects, we exposed mice to a low-fat (LF) or high-fat (HF) diet for only 5 days. The response to OxR was assessed by calorimetric measurements, followed by analysis of gene expression in liver and epididymal white adipose tissue (eWAT) and serum markers for e.g. protein glycation and oxidation. Although HF feeding increased body weight, HF and LF mice did not differ in indirect calorimetric values under normoxic conditions and in a fasting state. Exposure to OxR; however, increased oxygen consumption and lipid oxidation in HF mice versus LF mice. Furthermore, OxR induced gluconeogenesis and an antioxidant response in the liver of HF mice, whereas it induced de novo lipogenesis and an antioxidant response in eWAT of LF mice, indicating that HF and LF mice differed in their adaptation to OxR. OxR also increased serum markers of protein glycation and oxidation in HF mice, whereas these changes were absent in LF mice. Cumulatively, OxR is a promising new method to test food products on potential beneficial effects for human health.

Perinatal maternal high-fat consumption is known to increase the obesity and type 2 diabetes susceptibility and to impair exercise performance in the offspring. We hypothesize that epigenetic modifications in the skeletal muscle are partly responsible for this phenotype. To detect skeletal muscle genes affected by maternal nutrition, male offspring of low-fat (LF) and high-fat (HF) diet fed dams (BL6 mice) received LF diet upon weaning and were sacrificed at 6 or 25 weeks of age. Gene expression of Musculus quadriceps was investigated by microarray analysis revealing an up-regulation of the nuclear receptor Nr4a1 by maternal HF feeding. This was accompanied by promoter hypomethylation of CpG-1408 which correlated with increased Nr4a1 gene expression at both ages. Offspring voluntary exercise training (by supplying running wheels from 7 to 25 weeks of age) normalized Nr4a1 methylation and gene expression respectively, and ameliorated the negative effects of maternal HF feeding on insulin sensitivity. Overall, Nr4a1 gene expression in skeletal muscle correlated with higher insulin levels during an oral glucose tolerance test and could, therefore, be involved in programming type 2 diabetes susceptibility in offspring exposed to perinatal high fat diet.

Background: Body weight (BW) cycling, the yo-yo effect, is generally thought to have adverse effects on human metabolic health. However, human and animal experiments are limited in number and do not provide clear answers, partly due to large variations in experimental design, parameters measured, and definitions of BW cycling. Here, we examined the effect of repetitive BW cycling versus single- and non-cycling control groups, without alterations in diet composition, on steady state BW and metabolic parameters. Methods: We induced well-defined BW cycles on a semi-purified high fat diet in C57BL/6J mice, a well-described animal model for diet-induced obesity, and measured energy expenditure and relevant metabolic parameters. Results: Our setup indeed resulted in the intended BW changes and always reached a stage of energy balance. A history of weight cycling did not result in increased BW or fat mass compared with the control group, nor in deteriorated serum concentrations of glucose, adipokines and serum triglyceride and free fatty acid (FFA) concentrations. If anything, BW tended to be reduced, presumably because of a reduced overall energy intake in BW cycling animals. Conclusion: Repeated cycling in BW without changes in diet composition does not lead to impaired metabolic health nor increased BW (gain).

Transient neonatal 6-propyl-2-thiouracil (PTU) induced hypothyroidism affects Leydig and Sertoli cell numbers in the developing testis, resulting in increased adult testis size. The hypothyroid condition was thought to be responsible, an assumption questioned by studies showing that uninterrupted fetal/postnatal hypothyroidism did not affect adult testis size. Here, we investigated effects of transient hypothyroidism on Leydig and Sertoli cell development, employing a perinatal iodide-deficient diet in combination with sodium perchlorate. This hypothyroidism inducing diet was continued until days 1, 7, 14, or 28 postpartum (pp) respectively, when the rats were switched to a euthyroid diet and followed up to adulthood. Continuous euthyroid and hypothyroid, and neonatal PTU-treated rats switched to the euthyroid diet at 28 days pp, were included for comparison. No effects on formation of the adult-type Leydig cell population or on Sertoli cell proliferation and differentiation were observed when the diet switched at/or before day 14 pp. However, when the diet was discontinued at day 28 pp, Leydig cell development was delayed similarly to what was observed in chronic hypothyroid rats. Surprisingly, Sertoli cell proliferation was 6- to 8-fold increased 2 days after the diet switch and remained elevated the next days. In adulthood, Sertoli cell number per seminiferous tubule cross-section and consequently testis weight was increased in this group. These observations implicate that increased adult testis size in transiently hypothyroid rats is not caused by the hypothyroid condition per se, but originates from augmented Sertoli cell proliferation as a consequence of rapid normalization of thyroid hormone concentrations.

Absorption; Biodistribution; Genetic polymorphisms; Intestine; Macula lutea; International audience; Carotenoid dietary intake and their endogenous levels have been associated with a decreased risk of several chronic diseases. There are indications that carotenoid bioavailability depends, in addition to the food matrix, on host factors. These include diseases (e.g. colitis), life-style habits (e.g. smoking), gender and age, as well as genetic variations including single nucleotide polymorphisms that govern carotenoid metabolism. These are expected to explain interindividual differences that contribute to carotenoid uptake, distribution, metabolism and excretion, and therefore possibly also their association with disease risk. For instance, digestion enzymes fostering micellization (PNLIP, CES), expression of uptake/efflux transporters (SR-BI, CD36, NPC1L1), cleavage enzymes (BCO1/2), intracellular transporters (FABP2), secretion into chylomicrons (APOB, MTTP), carotenoid metabolism in the blood and liver (LPL, APO C/E, LDLR), and distribution to target tissues such as adipose tissue or macula (GSTP1, StARD3) depend on the activity of these proteins. In addition, human microbiota, e.g. via altering bile-acid concentrations, may play a role in carotenoid bioavailability. In order to comprehend individual, variable responses to these compounds, an improved knowledge on intra-/interindividual factors determining carotenoid bioavailability, including tissue distribution, is required. Here, we highlight the current knowledge on factors that may explain such intra-/interindividual differences.

Housing of laboratory mice at room temperature (22oC) might be considered a constant cold stress, which induces a thermogenic program in brown adipose tissue (BAT). However, the early adaptive response of white adipose tissue (WAT), the fat storage organ of the body, to a change from thermoneutrality to room temperature is not known. This was investigated here for various WAT depots, focusing on epididymal WAT (eWAT), widely used as reference depot. Male adult diet-induced obese (DIO) C57BL/6JOlaHsd mice housed at thermoneutrality (29oC), were for five days either switched to room temperature (22oC) or remained at thermoneutrality. Energy metabolism was continuously measured using indirect calorimetry. At the end of the study, serum metabolomics and WAT transcriptomics were performed. We confirmed activation of the thermogenic program in 22oC housed mice. Body weight and total fat mass were reduced. Whole body energy expenditure was increased, with a higher fatty acid to carbohydrate oxidation ratio and increased serum acylcarnitine levels, while energy intake was not significantly different between the two groups. Transcriptome analysis of eWAT identified tissue remodelling and inflammation as the most affected processes. Expression of pro-inflammatory M1 macrophage-related genes, and M1 over M2 macrophage ratio were decreased, which might be linked to an increased insulin sensitivity. Markers of thermogenesis were not altered in eWAT. Decreased expression of tryptophan hydroxylase 2 (Tph2) and cholecystokinin (Cck) might represent altered neuroendocrine signalling. eWAT itself does not show increased fatty acid oxidation. The three measured WATs, epididymal, mesenteric, and retroperitoneal, showed mainly similar responses; reduced inflammation (s100a8), decreased carbohydrate oxidation, and no or small differences in fatty acid oxidation. However, Ucp1 was only expressed and increased in rWAT in 22ᵒC housed mice. Cck expression was decreased in the three WATs, significantly in eWAT and rWAT, in contrast to Tph2, which was decreased in eWAT while not expressed in mWAT and rWAT. Our data show that tissue remodelling, inflammation and neuroendocrine signalling are early responses in WAT to a moderate decrease in environmental temperature.

Controlled studies on the effect of exercise on intestinal uptake of protein are scarce and underlying mechanisms largely unclear. We studied the uptake of the major allergen Ara h 6 following peanut consumption in an exercise model and compared this with changes in markers of intestinal permeability and integrity. Ten overnight-fasted healthy non-allergic men (n = 4) and women (n = 6) (23 +/- 4 years) ingested 100 g of peanuts together with a lactulose/rhamnose (L/R) solution, followed by rest or by 60 min cycling at 70% of their maximal workload. Significantly higher, though variable, levels of Ara h 6 in serum were found during exercise compared to rest (Peak p = 0.03; area under the curve p = 0.006), with individual fold changes ranging from no increase to an increase of over 150-fold in the uptake of Ara h 6. Similarly, uptake of lactulose (2-18 fold change, p = 0.0009) and L/R ratios (0.4-7.9 fold change, p = 0.04) were significantly increased which indicates an increase in intestinal permeability. Intestinal permeability and uptake of Ara h 6 were strongly correlated (r = 0.77, p

SummaryPerivascular adipose tissue (PVAT) surrounds blood vessels and releases paracrine factors, such as cytokines, which regulate local inflammation. The inflammatory state of PVAT has an important role in vascular disease; a pro-inflammatory state has been related with atherosclerosis development, whereas an anti-inflammatory one is protective. Cold exposure beneficially affects immune responses and, could thus impact the pathogenesis of cardiovascular diseases. In this study, we investigated the effects of one-week of cold exposure at 4°C of ferrets on aortic PVAT (aPVAT) versus subcutaneous adipose tissue. Ferrets were used because of the similarity of their adipose tissues to those of humans. A ferret-specific Agilent microarray was designed to cover the complete ferret genome and global gene expression analysis was performed. The data showed that cold exposure altered gene expression mainly in aPVAT. Most of the regulated genes were associated with cell cycle, immune response and gene expression regulation, and were mainly down-regulated. Regarding the effects on immune response, cold acclimation decreased the expression of genes involved in antigen recognition and presentation, cytokine signalling and immune system maturation and activation. This immunosuppressive gene expression pattern was depot-specific, as it was not observed in the inguinal subcutaneous depot. Interestingly, this depression in immune response related genes was also evident in peripheral blood mononuclear cells (PBMC). In conclusion, these results reveal that cold acclimation produces an inhibition of immune response-related pathways in aPVAT, reflected in PBMC, indicative of an anti-inflammatory response, which can potentially be exploited for the enhancement or maintenance of cardiovascular health.Supplementary Material to this article is available online at www.thrombosis-online.com.

In higher primates, development of the adult population of Leydig cells has received little attention. Here, the emergence of 3beta-hydroxysteroid dehydrogenase (HSD3B) positive cells in the testis of the rhesus monkey was examined during spontaneous puberty, and correlated with S-phase labeling in the interstitium at this critical stage of development. In addition, the relative role of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in initiating the pubertal expansion of Leydig cells was studied by precociously stimulating the juvenile testis in vivo with pulsatile 11-day infusions of recombinant LH and FSH, either alone or in combination. At the time of castration, testes were immersion fixed in Bouin's, embedded in paraffin, and sectioned at 5mum. Leydig cells/testis were enumerated using HSD3B as a Leydig cell marker. Leydig cell number per testis increased progressively during puberty to reach values in the adult approximately 10 fold greater than in early-pubertal animals. The rise in cell number was associated with an increase in nuclear diameter. That the pubertal expansion of Leydig cell number was driven primarily by the increase in LH secretion at this stage of development was suggested by the finding that precocious stimulation of mid-juvenile monkeys with LH, either alone or in combination with that of FSH, resulted in a 20-30 fold increase in the number of HSD3B-positive cells. Interestingly, precocious FSH stimulation, alone, also resulted in appearance of Leydig cells as indicated by the occasional HSD3B-positive cell in the interstitium. The nuclear diameter of these Leydig cells, however, was less than that of those generated in response to LH.

β-Carotene (BC) is omnipresent in our diet, both as natural food component as well as an additive. BC and its metabolites have important biological functions. For this reason, BC is generally considered to be a health promoting compound. Two human trials, however, have described adverse effects in lung tissue, increasing the risk of lung cancer. We previously applied transcriptomic analyses in a unique animal model, beta-carotene 15,15'-monooxygenase 1 knockout (Bcmo1(-/-)) mice that are, like humans, able to accumulate intact BC. In our search to unravel the molecular action of BC in the lung, we previously identified two genes particularly strongly down-regulated by BC in lung tissue of the male Bcmo1(-/-) mice: frizzled homologue 6 (Fzd6) and collagen triple helix repeat containing 1 (Cthrc1). In the present study, our aim was to further elucidate the role of FZD6 in lung epithelial cells and to provide a mechanistic explanation for BC increased lung cancer risk in humans. We performed whole genome microarray analysis on silenced FZD6 in non-tumor human type II bronchial epithelial BEAS-2B cells using RNAi. To directly link FZD6 to BC-effects on the lung, we compared the FZD6-silenced BEAS-2B gene expression profile to the BC-dependent gene expression profile of Bcmo1(-/-) mouse lungs. A number of relevant genes were regulated in the same direction in FZD6(-) BEAS-2B and in BC-exposed lungs of Bcmo1(-/-) mice and revealed enrichment of the Gene Ontology terms "oncogenes", "cell proliferation" and "cell cycle", which suggests a mediating role of FZD6 in BC-induced uncontrolled proliferation of lung cells.

Non-alcoholic fatty liver disease (NAFLD) accompanies obesity and insulin resistance. Recent meta-analysis suggested omega-3 polyunsaturated fatty acids DHA and EPA to decrease liver fat in NAFLD patients. Anti-inflammatory, hypolipidemic, and insulin-sensitizing effects of DHA/EPA depend on their lipid form, with marine phospholipids showing better efficacy than fish oils. We characterized the mechanisms underlying beneficial effects of DHA/EPA phospholipids, alone or combined with an antidiabetic drug, on hepatosteatosis. C57BL/6N mice were fed for 7 weeks an obesogenic high-fat diet (cHF) or cHF-based interventions: (i) cHF supplemented with phosphatidylcholine-rich concentrate from herring (replacing 10% of dietary lipids; PC), (ii) cHF containing rosiglitazone (10 mg/kg diet; R), or (iii) PC + R. Metabolic analyses, hepatic gene expression and lipidome profiling were performed. Results showed that PC and PC + R prevented cHF-induced weight gain and glucose intolerance, while all interventions reduced abdominal fat and plasma triacylglycerols. PC and PC + R also lowered hepatic and plasma cholesterol and reduced hepatosteatosis. Microarray analysis revealed integrated down-regulation of hepatic lipogenic and cholesterol biosynthesis pathways by PC, while R-induced lipogenesis was fully counteracted in PC + R. Gene expression changes in PC and PC + R were associated with preferential enrichment of hepatic phosphatidylcholine and phosphatidylethanolamine fractions by DHA/EPA. The complex down-regulation of hepatic lipogenic and cholesterol biosynthesis genes and the antisteatotic effects were unique to DHA/EPA-containing phospholipids, since they were absent in mice fed soy-derived phosphatidylcholine. Thus, inhibition of lipid and cholesterol biosynthesis associated with potent antisteatotic effects in the liver in response to DHA/EPA-containing phospholipids support their use in NAFLD prevention and treatment.

UCP1-Tg mice with ectopic expression of uncoupling protein 1 (UCP1) in skeletal muscle (SM) are a model of improved substrate metabolism and increased longevity. Analysis of myokine expression showed an induction of fibroblast growth factor 21 (FGF21) in SM, resulting in approximately fivefold elevated circulating FGF21 in UCP1-Tg mice. Despite a reduced muscle mass, UCP1-Tg mice showed no evidence for a myopathy or muscle autophagy deficiency but an activation of integrated stress response (ISR; eIF2α/ATF4) in SM. Targeting mitochondrial function in vitro by treating C2C12 myoblasts with the uncoupler FCCP resulted in a dose-dependent activation of ISR, which was associated with increased expression of FGF21, which was also observed by treatment with respiratory chain inhibitors antimycin A and myxothiazol. The cofactor required for FGF21 action, β-klotho, was expressed in white adipose tissue (WAT) of UCP1-Tg mice, which showed an increased browning of WAT similar to what occurred in altered adipocyte morphology, increased brown adipocyte markers (UCP1, CIDEA), lipolysis (HSL phosphorylation), and respiratory capacity. Importantly, treatment of primary white adipocytes with serum of transgenic mice resulted in increased UCP1 expression. Additionally, UCP1-Tg mice showed reduced body length through the suppressed IGF-I-GH axis and decreased bone mass. We conclude that the induction of FGF21 as a myokine is coupled to disturbance of mitochondrial function and ISR activation in SM. FGF21 released from SM has endocrine effects leading to increased browning of WAT and can explain the healthy metabolic phenotype of UCP1-Tg mice. These results confirm muscle as an important endocrine regulator of whole body metabolism.

Polyphenols generally show beneficial health effects upon supplementation in diet-induced obese rodent models, including reduced body weight gain and reduced levels of markers for cardiovascular diseases (CVD). However, there appear to be large differences between studies, which might be due to differences in models, strains, dietary background, or even concentration of polyphenol that is used. Therefore, we performed a systematic phenotypic evaluation of the effects of selected polyphenols in wildtype C57BL/6JOlaHsd mice. Epigallocatechin-gallate, quercetin, and resveratrol, representing three different phenolic classes, were each added in equimolar amounts (0.50% (w/w), 0.33%, and 0.25%, respectively) to a purified moderate high fat (30 energy%) diet for 12 weeks. We studied the polyphenol-induced physiological and molecular effects between them and relative to the non-supplemented control group during and at the end of the nutritional intervention. Results showed that these polyphenols were present in circulation, but did not induce beneficial health effects as analyzed by oral glucose tolerance testing or serum adipokines and CVD-markers such as vascular adhesion molecules. Remarkably, transcriptomics of white adipose tissue showed overlapping sets of significantly differential transcript levels between these polyphenols; AMPK and Notch signaling were affected by these polyphenols. However, mitochondrial processes and mitochondrial density in this tissue did not differ between the polyphenols, which suggested that there was no direct effect on adipose tissue.

Replacing part of glucose with galactose in the post-weaning diet beneficially affects later life metabolic health in female mice. The liver is the main site of galactose metabolism, but the direct effects of this dietary intervention on the liver in the post-weaning period are not known. The aim of this study was to elucidate this. Weanling female mice (C57BL/6JRccHsd) were fed a starch containing diet with glucose (32 en%) monosaccharide (GLU), or a diet with glucose and galactose (1:1 both 16 en%) (GLU+GAL). Body weight, body composition, and food intake were determined weekly. After 3 weeks, mice were sacrificed, and serum and liver tissues were collected. Global hepatic mRNA expression was analyzed and hepatic triglyceride (TG) and glycogen contents were determined by enzymatic assays. Body weight and body composition were similar in both groups, despite higher food intake in mice on GLU+GAL diet. Hepatic TG content was lower in GLU+GAL-fed than GLU-fed females, while glycogen levels were unaffected. Analysis of global expression patterns of hepatic mRNA showed that mainly inflammation-related pathways were affected by the diet, which were predominantly downregulated in GLU+GAL-fed females compared to GLU-fed females. This reduction in inflammation in GLU+GAL-fed females was also reflected by decreased serum concentrations of acute phase protein Serum amyloid A 3. In conclusion, replacing part of glucose with galactose in the post-weaning diet reduces hepatic TG content and hepatic inflammation.

Obesity is associated with white adipose tissue (WAT) hypoxia and inflammation. We aimed to test whether mild environmental oxygen restriction (OxR, 13% O2), imposing tissue hypoxia, triggers WAT inflammation in obese mice. Thirteen weeks diet-induced obese male adult C57BL/6JOlaHsd mice housed at thermoneutrality were exposed for five days to OxR versus normoxia. WAT and blood were isolated and used for analysis of metabolites and adipokines, WAT histology and macrophage staining, and WAT transcriptomics. OxR increased circulating levels of haemoglobin and haematocrit as well as hypoxia responsive transcripts in WAT and decreased blood glucose, indicating systemic and tissue hypoxia. WAT aconitase activity was inhibited. Macrophage infiltration as marker for WAT inflammation tended to be decreased, which was supported by down regulation of inflammatory genes S100a8, Ccl8, Clec9a, Saa3, Mgst2, and Saa1. Other down regulated processes include cytoskeleton remodelling and metabolism, while response to hypoxia appeared most prominently up regulated. The adipokines coiled-coil domain containing 3 (CCDC3) and adiponectin, as well as the putative WAT hormone cholecystokinin (CCK), were reduced by OxR on transcript (Cck, Ccdc3) and/or serum protein level (adiponectin, CCDC3). Conclusively, our data demonstrate that also in obese mice OxR does not trigger WAT inflammation. However, OxR does evoke a metabolic response in WAT, with CCDC3 and adiponectin as potential markers for systemic or WAT hypoxia.

Using standardized, semipurified diets is a crucial factor for reproducibility of experimental nutritional studies. For the purpose of comparability and integration of research, two European consortia, Mitofood and BIOCLAIMS, proposed an AIN-93-based standard reference diet, the standardized BIOCLAIMS low-fat diet (LFD) as well as a high-fat diet (HFD). In order to evaluate the BIOCLAIMS LFD and HFD, we performed short-term (5 days) and long-term (12 weeks) feeding experiments using male C57BL/6 mice. The HFD has the same composition as the LFD except the fat content is increased to 40% energy in exchange for carbohydrates. Both diets were accepted by the animals and proof of principle was given that the BIOCLAIMS HFD increases body weight and body fat and affects glucose homeostasis. Short-term feeding trials (5 days) were performed in order to identify metabolic and molecular parameters which can serve as acute predictors for metabolic disorders due to high-fat diet-induced obesity. We analyzed gene expression in gonadal white adipose tissue of short- and long-term fed animals with whole genome microarrays. The BIOCLAIMS HFD strongly influenced gene expression in white adipose tissue after short- and long-term intervention. A total number of 973 and 4678 transcripts were significantly different between both diets after 5 days feeding and 12 weeks feeding, respectively. A total number of 764 transcripts encoding 549 genes were significantly differentially regulated between LF and HF animals after 12 weeks feeding as well as after 5 days feeding. Of these 549 overlapping genes, a substantial number (434 genes) were expressed at a lower level and 115 genes were expressed at a higher level in the HF mice compared to the LF mice. Without exception, all genes were regulated equally. Pathway analysis revealed a prominent role for genes involved in lipid metabolism, carbohydrate metabolism and oxidative phosphorylation. This was confirmed by quantitative real-time reverse transcription PCR. The high predictive value of gene expression changes in our short-term study compared to long-term high fat feeding is a promising step to get well-defined, early biomarkers that could shorten animal trials considerably and allow a more rapid and efficient screening of different compounds.

Understanding body weight regulation is essential to fight obesity. Mouse studies, using different types of diets, showed conflicting results in terms of body weight persistence after changing from an ad libitum high-fat diet to an ad libitum low-fat diet. In this study, we questioned specifically whether the energy content of the diet has a lasting effect on energy balance and body weight, using multiple switches and two purified diets with a different fat-to-sugar ratio, but otherwise identical ingredients. Young-adult obesity-prone male C57BL/6J mice were fed single or double switches of semi-purified diets with either 10 energy % (en%) fat (LF) or 40en% fat (HF), with starch replaced by fat, while protein content remained equal. After none, one or two dietary changes, energy metabolism was assessed at 5, 14 and 19 weeks. We observed no systematic continuous compensation in diet and energy intake when returning to LF after HF consumption. Body weight, white adipose tissue mass and histology, serum metabolic parameters, energy expenditure and substrate usage all significantly reflected the current diet intake, independent of dietary changes. This contrasts with studies that used diets with different ingredients and showed persistent effects of dietary history on body weight, suggesting diet-dependent metabolic set points. We conclude that body weight and metabolic parameters 'settle', based on current energetic input and output. This study also highlights the importance of considering the choice of diet in physiological and metabolic intervention studies.

cope Metabolic flexibility is the ability to switch metabolism between carbohydrate oxidation (CHO) and fatty acid oxidation (FAO) and is a biomarker for metabolic health. The effect on metabolic health of nicotinamide riboside (NR) as an exclusive source of vitamin B3 is unknown and is examined here for a wide range of NR. Design and methods Nine-week-old male C57BL/6JRcc mice received a semi-purified mildly obesogenic (40 en% fat) diet containing 0.14% L-tryptophan and either 5, 15, 30, 180, or 900 mg NR per kg diet for 15 weeks. Body composition and metabolic parameters were analyzed. Metabolic flexibility was measured using indirect calorimetry. Gene expression in epididymal white adipose tissue (eWAT) was measured using qRT-PCR . Results The maximum delta respiratory exchange ratio when switching from CHO to FAO (maxΔRERCHO1→FAO) and when switching from FAO to CHO (maxΔRERFAO→CHO2) were largest in 30 mg NR per kg diet (30NR). In eWAT, the gene expression of Pparγ, a master regulator of adipogenesis, and of Sod2 and Prdx3, two antioxidant genes, were significantly upregulated in 30NR compared to 5NR. Conclusion 30NR is most beneficial for metabolic health, in terms of metabolic flexibility and eWAT gene expression, of mice on an obesogenic diet.

Some studies have suggested that allergens may appear in the circulation after ingestion of allergenic food sources. The reported levels of allergen in serum, however, are low, and conclusions between studies differ. Here, we investigated factors that determine the detection of allergens in serum after consumption of peanuts. Ten healthy volunteers ingested 100g of light-roasted peanuts. Serum samples were taken at regular intervals for six hours. A double monoclonal sandwich ELISA was used to analyse the presence and quantity of the major peanut allergen Ara h 6 in serum. In 4 out of 10 subjects, no Ara h 6 could be detected. Purified Ara h 6 that was digested in vitro was still reactive in the ELISA, rejecting the possibility that digestion leads to small peptides that could not be detected. Spiking of purified Ara h 6 in baseline serum showed that the pre-ingestion serum of these four subjects partially prevented Ara h 6 to react in the ELISA, with a reduction of reactivity of up to 3 orders of magnitude or more. Pre-ingestion serum of the other six subjects did not show such an effect. The reduction of reactivity of Ara h 6 coincided with high titres of IgG and IgG4, and removal of IgG from pre-ingestion serum abolished this effect completely, indicating that IgG and IgG4 inhibited the reactivity of Ara h6 in the ELISA. We conclude that some individuals have IgG and IgG4 against food allergens in their blood, which interferes with detection of such food allergens in serum. Because this effect does not occur for each individual, the possibility of such interference should be taken into consideration when interpreting immunochemical studies on the absorption of food allergens in serum.

What is the central question of this study? Exercise is known to induce stress-related physiological responses, such as changes in intestinal barrier function. Our aim was to determine the test-retest repeatability of these responses in well-trained individuals. What is the main finding and its importance? Responses to strenuous exercise, as indicated by stress-related markers such as intestinal integrity markers and myokines, showed high test-retest variation. Even in well-trained young men an adapted response is seen after a single repetition after 1 week. This finding has implications for the design of studies aimed at evaluating physiological responses to exercise. Strenuous exercise induces different stress-related physiological changes, potentially including changes in intestinal barrier function. In the Protégé Study (ISRCTN14236739; www.isrctn.com), we determined the test-retest repeatability in responses to exercise in well-trained individuals. Eleven well-trained men (27 ± 4 years old) completed an exercise protocol that consisted of intensive cycling intervals, followed by an overnight fast and an additional 90 min cycling phase at 50% of maximal workload the next morning. The day before (rest), and immediately after the exercise protocol (exercise) a lactulose and rhamnose solution was ingested. Markers of energy metabolism, lactulose-to-rhamnose ratio, several cytokines and potential stress-related markers were measured at rest and during exercise. In addition, untargeted urine metabolite profiles were obtained. The complete procedure (Test) was repeated 1 week later (Retest) to assess repeatability. Metabolic effect parameters with regard to energy metabolism and urine metabolomics were similar for both the Test and Retest period, underlining comparable exercise load. Following exercise, intestinal permeability (1 h plasma lactulose-to-rhamnose ratio) and the serum interleukin-6, interleukin-10, fibroblast growth factor-21 and muscle creatine kinase concentrations were significantly increased compared with rest only during the first test and not when the test was repeated. Responses to strenuous exercise in well-trained young men, as indicated by intestinal markers and myokines, show adaptation in Test-Retest outcome. This might be attributable to a carry-over effect of the defense mechanisms triggered during the Test. This finding has implications for the design of studies aimed at evaluating physiological responses to exercise.

Intake of high-protein (HP) diets has increased over the last years, mainly due to their popularity for body weight control. Liver is the main organ handling ingested macronutrients and it is associated with the beginning of different pathologies. We aimed to deepen our knowledge on molecular pathways affected by long-term intake of an HP diet. We performed a transcriptome analysis on liver of rats chronically fed with a casein-rich HP diet and analyzed molecular parameters related to liver injury. Chronic increase in the dietary protein/carbohydrate ratio up-regulated processes related with amino acid uptake/metabolism and lipid synthesis, promoting a molecular environment indicative of hepatic triacylglycerol (TG) deposition. Moreover, changes in expression of genes involved in acid–base maintenance and oxidative stress indicate alterations in the pH balance due to the high acid load of the diet, which has been linked to liver/health damage. Up-regulation of immune-related genes was also observed. In concordance with changes at gene expression level, we observed increased liver TG content and increased serum markers of hepatic injury/inflammation (aspartate transaminase, C-reactive protein and TNF-alpha). Moreover, the HP diet strongly increased hepatic mRNA and protein levels of HSP90, a marker of liver injury. Thus, we show for the first time that long-term consumption of an HP diet, resulting in a high acid load, results in a hepatic transcriptome signature reflecting increased TG deposition and increased signs of health risk (increased inflammation, alterations in the acid–base equilibrium and oxidative stress). Persistence of this altered metabolic status could have unhealthy consequences.

The long-term effects of chronic hypothyroidism on ovarian follicular development in adulthood are not well known. Using a rat model of chronic diet-induced hypothyroidism initiated in the fetal period, we investigated the effects of prolonged reduced plasma thyroid hormone concentrations on the ovarian follicular reserve and ovulation rate in prepubertal (12-day-old) and adult (64-day-old and 120-day-old) rats. Besides, antioxidant gene expression, mitochondrial density and the occurrence of oxidative stress were analyzed. Our results show that continuous hypothyroidism results in lower preantral and antral follicle numbers in adulthood, accompanied by a higher percentage of atretic follicles, when compared to euthyroid age-matched controls. Not surprisingly, ovulation rate was lower in the hypothyroid rats. At the age of 120 days, the mRNA and protein content of superoxide dismutase 1 (SOD1) were significantly increased while catalase (CAT) mRNA and protein content was significantly decreased, suggesting a disturbed antioxidant defense capacity of ovarian cells in the hypothyroid animals. This was supported by a significant reduction in the expression of peroxiredoxin 3 ( ITALIC! Prdx3), thioredoxin reductase 1 ( ITALIC! Txnrd1), and uncoupling protein 2 ( ITALIC! Ucp2) and a downward trend in glutathione peroxidase 3 ( ITALIC! Gpx3) and glutathione S-transferase mu 2 ( ITALIC! Gstm2) expression. These changes in gene expression were likely responsible for the increased immunostaining of the oxidative stress marker 4-hydroxynonenal. Together these results suggest that chronic hypothyroidism initiated in the fetal/neonatal period results in a decreased ovulation rate associated with a disturbance of the antioxidant defense system in the ovary.

Background/Objectives: Consumption of fat-rich foods is associated with obesity and related alterations. However, there is a group of individuals, the metabolically obese normal-weight (MONW) subjects, who present normal body weight but have metabolic features characteristic of the obese status, including fat deposition in critical tissues such as liver, recognized as a major cause for the promotion of metabolic diseases. Our aim was to better understand metabolic alterations present in liver of MONW rats applying whole genome transcriptome analysis. Methods: Wistar rats were chronically fed a high-fat diet isocaloric relative to Control animals to avoid the hyperphagia and overweight and to mimic MONW features. Liver transcriptome analysis of both groups was performed. Results: Sustained intake of an isocaloric high-fat diet had a deep impact on the liver transcriptome, mainly affecting lipid metabolism. Although serum cholesterol levels were not affected, circulating triacylglycerols were lower, and metabolic adaptations at gene expression level indicated adaptation toward handling the increased fat content of the diet, an increased triacylglycerol and cholesterol deposition in liver of MONW rats was observed. Moreover, gene expression pointed to increased risk of liver injury. One of the top upregulated genes in this tissue was Krt23, a marker of hepatic disease in humans that was also increased at the protein level.Conclusion:Long-term intake of a high-fat diet, even in the absence of overweight/obesity or increase in classical blood risk biomarkers, promotes a molecular environment leading to hepatic lipid accumulation and increasing the risk of suffering from hepatic diseases.

Moderate selenium deficiency may lead to an impaired capacity to cope with health challenges. Functional effects of suboptimal selenium intake are not fully known, and biomarkers for an insufficient selenium supply are inadequate. We therefore fed mice diets of moderately deficient or adequate selenium intake for 6 weeks. Changes in global gene expression were monitored by microarray analysis in splenic leukocytes. Genes for four selenoproteins, Sepw1, Gpx1, Selh and Sep15, were the most significantly down-regulated in moderate selenium deficiency, and this was confirmed by quantitative polymerase chain reaction (qPCR). Classification of significantly affected genes revealed that processes related to inflammation, heme biosynthesis, DNA replication and transcription, cell cycle and transport were affected by selenium restriction. Down-regulation by moderate selenium deficiency of specific genes involved in inflammation and heme biosynthesis was confirmed by qPCR. Myeloperoxidase and lysozyme activities were decreased in selenium-restricted leukocytes, providing evidence for functional consequences. Genes for 31 nuclear factor (NF)-kappaB targets were down-regulated in moderate selenium deficiency, indicating an impaired NF-kappaB signaling. Together, the observed changes point to a disturbance in inflammatory response. The selenoproteins found here to be sensitive to selenium intake in murine leukocytes might also be useful as biomarkers for a moderate selenium deficiency in humans.

Experimental replication is fundamental for practicing science. To reduce variability, it is essential to control sources of variation as much as possible. Diet is an important factor that can influence many processes and functional outcomes in studies performed with rodent models. This is especially true for, but not limited to, nutritional studies. To compare functional effects of different nutrients, it is important to use standardized, semi-purified diets. Here, we propose and describe a standard reference diet, the BIOCLAIMS standard diet. The diet is AIN-93 based, but further defined with dietary and experimental requirements taken into account that allow for experiments with bioactive food components and natural (non-expensive) labeling. This diet will be implemented by two European research consortia, Mitofood and BIOCLAIMS, to ensure inter-laboratory comparability.

Flavonoids are bioactive food compounds with potential lipid-lowering effects. Commercially available enzymatic assays are widely used to determine free fatty acid (FFA) and triglyceride (TG) levels both in vivo in plasma or serum and in vitro in cell culture medium or cell lysate. However, we have observed that various flavonoids interfere with peroxidases used in these enzymatic assays, resulting in incorrect lower FFA and TG levels than actually present. Furthermore, addition of isorhamnetin or the major metabolite of the flavonoid quercetin in human and rat plasma, quercetin-3-O-glucuronide, to murine serum also resulted in a significant reduction of the detected TG levels, while a trend was seen for FFA levels. It is concluded that when applying these assays, vigilance is needed and alternative analytical methods, directly assessing FFA or TG levels, should be used for studying the biological effects of flavonoids on FFA and TG levels. Figure Interference of different flavonoids spiked into cell culture medium on the FFA (A) and TG (B) levels, and interference of quercetin (Q) and its major metabolite, quercetin-3-O-glucuronide (Q3G), and isorhamnetin (I) spiked into murine serum on the FFA (C) and TG (D) levels, as measured by enzymatic assays. Flavonoids analysed are quercetin (Q), (+)-catechin (C), luteolin (L), genistein (G), naringenin (N), and kaempferol (K). Asterisk indicates a significant difference (p

Molecular mechanisms triggered by high dietary beta-carotene (BC) intake in lung are largely unknown. We performed microarray gene expression analysis on lung tissue of BC supplemented beta-carotene 15,15'-monooxygenase 1 knockout (Bcmo1 (-/-)) mice, which are-like humans-able to accumulate BC. Our main observation was that the genes were regulated in an opposite direction in male and female Bcmo1 (-/-) mice by BC. The steroid biosynthetic pathway was overrepresented in BC-supplemented male Bcmo1 (-/-) mice. Testosterone levels were higher after BC supplementation only in Bcmo1 (-/-) mice, which had, unlike wild-type (Bcmo1 (+/+)) mice, large variations. We hypothesize that BC possibly affects hormone synthesis or metabolism. Since sex hormones influence lung cancer risk, these data might contribute to an explanation for the previously found increased lung cancer risk after BC supplementation (ATBC and CARET studies). Moreover, effects of BC may depend on the presence of frequent human BCMO1 polymorphisms, since these effects were not found in wild-type mice.

An ongoing controversy exists on beneficial versus harmful effects of high beta-carotene (BC) intake, especially for the lung. To elucidate potential mechanisms, we studied effects of BC on lung gene expression. We used a beta-carotene 15,15'-monooxygenase 1 (Bcmo1) knockout mouse (Bcmo1(-/-)) model, unable to convert BC to retinoids, and wild-type mice (Bcmo1(+/+)) mice to dissect the effects of intact BC from effects of BC metabolites. As expected, BC supplementation resulted in a higher BC accumulation in lungs of Bcmo1(-/-) mice than in lungs of Bcmo1(+/+) mice. Whole mouse genome transcriptome analysis on lung tissue revealed that more genes were regulated in Bcmo1(-/-) mice than Bcmo1(+/+) mice upon BC supplementation. Frizzled homolog 6 (Fzd6) and collagen triple helix repeat containing 1 (Cthrc1) were significantly downregulated (fold changes -2.99 and -2.60, respectively, false discovery rate0.05) by BC in Bcmo1(-/-). Moreover, many olfactory receptors and many members of the protocadherin family were upregulated. Since both olfactory receptors and protocadherins have an important function in sensory nerves and Fzd6 and Cthrc1 are important in stem cell development, we hypothesize that BC might have an effect on the highly innervated pulmonary neuroendocrine cell (PNEC) cluster. PNECs are highly associated with sensory nerves and are important cells in the control of stem cells. A role for BC in the innervated PNEC cluster might be of particular importance in smoke-induced carcinogenesis since PNEC-derived lung cancer is highly associated with tobacco smoke.

Beta-carotene 15,15'-monooxygenase 1 knockout (Bcmo1 (-/-)) mice accumulate beta-carotene (BC) similarly to humans, whereas wild-type (Bcmo1 (+/+)) mice efficiently cleave BC. Bcmo1 (-/-) mice are therefore suitable to investigate BC-induced alterations in gene expression in lung, assessed by microarray analysis. Bcmo1 (-/-) mice receiving control diet had increased expression of inflammatory genes as compared to BC-supplemented Bcmo1 (-/-) mice and Bcmo1 (+/+) mice that received either control or BC-supplemented diets. Differential gene expression in Bcmo1 (-/-) mice was confirmed by real-time quantitative PCR. Histochemical analysis indeed showed an increase in inflammatory cells in lungs of control Bcmo1 (-/-) mice. Supported by metabolite and gene-expression data, we hypothesize that the increased inflammatory response is due to an altered BC metabolism, resulting in an increased vitamin A requirement in Bcmo1 (-/-) mice. This suggests that effects of BC may depend on inter-individual variations in BC-metabolizing enzymes, such as the frequently occurring human polymorphisms in BCMO1.

Flavonoids are inactivated by phase II metabolism and occur in the body as glucuronides. Mammalian beta-glucuronidase released from neutrophils at inflammatory sites may be able to deconjugate and thus activate flavonoid glucuronides. We have studied deconjugation kinetics and pH optimum for four sources of beta-glucuronidase (human neutrophil, human recombinant, myeloid PLB-985 cells, Helix pomatia) with five flavonoid glucuronides (quercetin-3-glucuronide, quercetin-3'-glucuronide, quercetin-4'-glucuronide, quercetin-7-glucuronide, 3'-methylquercetin-3-glucuronide), 4-methylumbelliferyl-beta-D-glucuronide, and para-nitrophenol-glucuronide. All substrate-enzyme combinations tested exhibited first order kinetics. The optimum pH for hydrolysis was between 3.5-5, with appreciable hydrolysis activities up to pH 5.5. At pH 4, the K(m) ranged 44-fold from 22 microM for quercetin-4'-glucuronide with Helix pomatia beta-glucuronidase, to 981 microM for para-nitrophenol-glucuronide with recombinant beta-glucuronidase. V(max) (range: 0.735-24.012 micromol x min(-1) x unit(-1) [1 unit is defined as the release of 1 microM 4-methylumbelliferyl-beta-D-glucuronide per min]) and the reaction rate constants at low substrate concentrations (k) (range: 0.002-0.062 min(-1) x (unit/L)(-1) were similar for all substrates-enzyme combinations tested. In conclusion, we show that beta-glucuronidase from four different sources, including human neutrophils, is able to deconjugate flavonoid glucuronides and non-flavonoid substrates at fairly similar kinetic rates. At inflammatory sites in vivo the pH, neutrophil and flavonoid glucuronide concentrations seem favorable for deconjugation. However, it remains to be confirmed whether this is actually the case.

Item does not contain fulltext We have shown in several controlled rat and human infection studies that dietary calcium improves intestinal resistance and strengthens the mucosal barrier. Reinforcement of gut barrier function may alleviate inflammatory bowel disease (IBD). Therefore, we investigated the effect of supplemental calcium on spontaneous colitis development in an experimental rat model of IBD. HLA-B27 transgenic rats were fed a purified high-fat diet containing either a low or high calcium concentration (30 and 120 mmol CaHPO4/kg diet, respectively) for almost 7 wk. Inert chromium EDTA (CrEDTA) was added to the diets to quantify intestinal permeability by measuring urinary CrEDTA excretion. Relative fecal wet weight was determined to quantify diarrhea. Colonic inflammation was determined histologically and by measuring mucosal interleukin (IL)-1beta. In addition, colonic mucosal gene expression of individual rats was analyzed using whole-genome microarrays. The calcium diet significantly inhibited the increase in intestinal permeability and diarrhea with time in HLA-B27 rats developing colitis compared with the control transgenic rats. Mucosal IL-1beta levels were lower in calcium-fed rats and histological colitis scores tended to be lower (P = 0.08). Supplemental calcium prevented the colitis-induced increase in the expression of extracellular matrix remodeling genes (e.g. matrix metalloproteinases, procollagens, and fibronectin), which was confirmed by quantitative real-time PCR and gelatin zymography. In conclusion, dietary calcium ameliorates several important aspects of colitis severity in HLA-B27 transgenic rats. Reduction of mucosal irritation by luminal components might be part of the mechanism. These results show promise for supplemental calcium as effective adjunct therapy for IBD.

A primary goal of nutrition research is to optimize health and prevent or delay disease. Biomarkers to quantify health optimization are needed since many if not most biomarkers are developed for diseases. Quantifying "normal homeostasis" and developing validated biomarkers are formidable tasks because of the robustness of homeostasis and of inter-individual diversity. In this paper, we discuss the science, strategies, and technologies for measuring parameters that define individual health. The following concepts are central to define the physiology of the healthy individual: (i) responses to a challenge of homeostasis will be more informative than static homeostatic measures; (ii) processes involved in maintaining homeostasis usually are multi-factorial and require quantitative analyses of the many individual components involved; (iii) health includes a large variation in "normality" and the effects of nutritional interventions may remain hidden in this "diversity of robustness," if incompletely analyzed. Specifically, comprehensive multi-parameter ("omics") analysis may identify key parameters (biomarkers) and lead to a greater understanding of health supporting processes. Perturbation tests that accurately target aspects of the overarching drivers of health (metabolism, oxidation, inflammation, and psychological stress) may be instrumental in creating knowledge for maintaining health and preventing disease through nutrition. © 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Chronic prenatally induced dietary hypothyroidism delays adult-type Leydig cell development, but does not block this process. Using a chemical model to induce hypothyroidism, it was suggested that development of a new population of Leydig cells was completely inhibited following the addition of the cytotoxic compound ethane-1,2-dimethyl sulphonate (EDS). In this study, we used a dietary approach to induce hypothyroidism and reinvestigated the regeneration of the Leydig cell population following EDS administration. Eighty-four day old euthyroid and chronically hypothyroid rats received an injection of EDS and were killed directly before or at regular intervals up to 77 days after EDS. In some control and hypothyroid animals, the first progenitor-type Leydig cells were observed at day 12 after EDS. At day 16, Leydig cell progenitors were present in all rats. The percentage of proliferating Leydig cells peaked in the euthyroid animals at day 21 after EDS. In the hypothyroid testis such a peak was not observed, although the percentage of proliferating regenerating Leydig cells was significantly higher from days 35 to 56 compared with the controls. This suggested that the wave of Leydig cell proliferation was delayed in the hypothyroid animals as compared with the euthyroid controls. On the day of EDS injection, the Leydig/Sertoli cell ratio was 37% lower in the hypothyroid rats compared with the controls. The Leydig/Sertoli cell ratio remained lower in the EDS-treated hypothyroid animals compared with the controls at all time points investigated. At day 77 after EDS, the Leydig cell population had returned to its pre-treatment size in both groups. Plasma testosterone production was reduced to below detectable levels immediately after EDS injection, and started to increase again on day 16, reaching pre-treatment values on day 21 in both groups. Taken together, severely reduced thyroid hormone levels did not block the regeneration of the adult-type Leydig cell population following EDS, as has been suggested previously.

Background Microbial infections induce ileal pancreatitis-associated protein/regenerating gene III (PAP/RegIII) mRNA expression. Despite increasing interest, little is known about the PAP/RegIII protein. Therefore, ileal mucosal PAP/RegIII protein expression, localization, and fecal excretion were studied in rats upon Salmonella infection. Results Salmonella infection increased ileal mucosal PAP/RegIII protein levels in enterocytes located at the crypt-villus junction. Increased colonization and translocation of Salmonella was associated with higher ileal mucosal PAP/RegIII levels and secretion of this protein in feces. Conclusions PAP/RegIII protein is increased in enterocytes of the ileal mucosa during Salmonella infection and is associated with infection severity. PAP/RegIII is excreted in feces and might be used as a new and non-invasive infection marker

An important part of the comparative approach to assess the safety of new crop plant varieties is an extensive compositional analysis, including the measurement of all key nutrients and antinutrients in a specific crop. The study described here investigates the applicability of `omics' technologies, transcriptomics and proteomics, as additional tools in this comparative safety assessment. The aim of the work was to assess the extent of the natural variation in ripening tomato fruits as a model crop and to determine whether it is possible to develop simple `ripening stage' criteria for the sampling of fruits for `omics' analyses. It is shown that the set-up of an `omics' study is of crucial importance. Samples under scrutiny should be well-matched with relation to environmental conditions during growth and harvest, including the stage of ripening, as is stipulated in international guidance documents for the nutritional and toxicological assessment of genetically modified plants.

The second generation of genetically modified (GM) plants that are moving towards the market are characterized by modifications that may be more complex and traits that more often are to the benefit of the consumer. These developments will have implications for the safety assessment of the resulting plant products. In part of the cases the same crop plant can, however, also be obtained by `conventional¿ breeding strategies. The breeder will decide on a case-by-case basis what will be the best strategy to reach the set target and whether genetic modification will form part of this strategy. This article discusses important aspects of the safety assessment of complex products derived from newly bred plant varieties obtained by different breeding strategies. On the basis of this overview, we conclude that the current process of the safety evaluation of GM versus conventionally bred plants is not well balanced. GM varieties are elaborately assessed, yet at the same time other crop plants resulting from conventional breeding strategies may warrant further food safety assessment for the benefit of the consumer. We propose to develop a general screening frame for all newly developed plant varieties to select varieties that cannot, on the basis of scientific criteria, be considered as safe as plant varieties that are already on the market.

Purpose of review Adipose tissue is an essential, highly dynamic and metabolically active tissue that vigorously communicates to support its primary function: the storage of lipids. It performs this function to secure energy supply and prevent lipotoxicity. Adipose tissue is essential for maintaining a healthy glucose and lipid homeostasis and failure results in disease. This review discusses causes of adipose tissue failure and four categories of bioactive food components that may help to prevent this. Recent findings Based on recent findings, it is argued that initial adipose failure following long-term excess energy intake may be the result of reduced mitochondrial capacity associated with altered mitochondrial reactive oxygen species signaling and adipose tissue hypoxia. Current data suggest that different classes of bioactive food components, including vitamin B3, retinoids, fatty acids and polyphenols, may have the potential to modulate mitochondrial function and consequently prevent adipose dysfunction in obesity. Summary It seems most attractive to aim nutritional intervention at the prevention of initial adipose dysfunction and hence to target dietary intervention at improvement of mitochondrial function.

Folate, a water-soluble B vitamin, is a cofactor in one-carbon metabolism and is essential for DNA synthesis, amino acid interconversion, methylation and, consequently, normal cell growth. In animals with existing pre-neoplastic and neoplastic lesions, folic acid supplementation increases the tumour burden. To identify processes that are affected by increased folic acid levels, we compared HT29 human colon cancer cells exposed to a chronic supplemental (100 ng/ml) level of folic acid to cells exposed to a normal (10 ng/ml) level of folic acid, in the presence of vitamin B12and other micronutrients involved in the folate–methionine cycle. In addition to higher intracellular folate levels, HT29 cells at 100 ng folic acid/ml displayed faster growth and higher metabolic activity. cDNA microarray analysis indicated an effect on cell turnover and Fe metabolism. We fully confirmed these effects at the physiological level. At 100 ng/ml, cell assays showed higher proliferation and apoptosis, while gene expression analysis and a lower E-cadherin protein expression indicated decreased differentiation. These results are in agreement with the promoting effect of folic acid supplementation on established colorectal neoplasms. The lower expression of genes related to Fe metabolism at 100 ng folic acid/ml was confirmed by lower intracellular Fe levels in the cells exposed to folic acid at 100 ng/ml. This suggests an effect of folate on Fe metabolism.

In whole genome microarray studies major gene expression changes are easily identified, but it is a challenge to capture small, but biologically important, changes. Pathway-based programs can capture small effects but may have the disadvantage of being restricted to functionally annotated genes. A structured approach toward the identification of major and small changes for interpretation of biological effects is needed. We present a structured approach, a framework, that addresses different considerations in 1) the identification of informative genes in microarray data sets and 2) the interpretation of their biological relevance. The steps of this framework include gene ranking, gene selection, gene grouping, and biological interpretation. Random forests (RF), which takes gene-gene interactions into account, is examined to rank and select genes. For human, mouse, and rat whole genome arrays, less than half of the probes on the array are annotated. Consequently, pathway analysis tools ignore half of the information present in the microarray data set. The framework described takes all genes into account. RF is a useful tool to rank genes by taking interactions into account. Applying a permutation approach, we were able to define an objective threshold for gene selection. RF combined with self-organizing maps identified genes with coordinated but small gene expression responses that were not fully annotated but corresponded to the same biological process. The presented approach provides a flexible framework for biological interpretation of microarray data sets. It includes all genes in the data set, takes gene-gene interactions into account, and provides an objective threshold for gene selection.

To be able to perform a comprehensive and rigorous benefit-risk analysis of individual food components, and of foods, a number of fundamental questions need to be addressed first. These include whether it is feasible to detect all relevant biological effects of foods and individual food components, how such effects can confidently be categorised into benefits and risks in relation to health and, for that matter, how health can be quantified. This article examines the last of these issues, focusing upon concepts for the development of new biomarkers of health. Clearly, there is scope for refinement of classical biomarkers so that they may be used to detect even earlier signs of disease, but this approach defines health solely as the absence of detectable disease or disease risk. We suggest that the health of a biological system may better be reflected by its ability to withstand and manage relevant physiological challenges so that homeostasis is maintained. We discuss the potential for expanding the range of current challenge tests for use in conjunction with functional genomic technologies to develop new types of biomarkers of health.

Item does not contain fulltext Mucosal pentraxin (Mptx), identified in rats, is a short pentraxin of unknown function. Other subfamily members are Serum amyloid P component (SAP), C-reactive protein (CRP) and Jeltraxin. Rat Mptx mRNA is predominantly expressed in colon and in vivo is strongly (30-fold) regulated by dietary heme and calcium, modulators of colon cancer risk. This renders Mptx a potential nutrient sensitive biomarker of gut health. To support a role as biomarker, we examined whether the pentraxin protein structure is conserved, whether Mptx protein is nutrient-sensitively expressed and whether Mptx is expressed in mouse and human. Sequence comparison and 3D modelling showed that rat Mptx is highly homologous to the other pentraxins. The calcium-binding site and subunit interaction sites are highly conserved, while a loop deletion and charged residues contribute to a distinctive �top� face of the pentamer. In accordance with mRNA expression, Mptx protein is strongly down-regulated in rat colon mucosa in response to high dietary heme intake. Mptx mRNA is expressed in rat and mouse colon, but not in human colon. A stop codon at the beginning of human exon two indicates loss of function, which may be related to differences in intestinal cell turnover between man and rodents.