In this study, we chose a carnivorous fish, turbot (Scophthalmus maximus L.), to examine its nutrient-sensing and metabolic responses after ingestion of diets with fishmeal (FM), or 45 % of FM replaced by soyabean meal (34·6 % dry diet) balanced with or without essential amino acids (EAA) to match the amino acid profile of FM diet for 30 d. After a 1-month feeding trial, fish growth, feed efficiency and nutrient retention were markedly reduced by soyabean meal-incorporated (SMI) diets. Compared with the FM diet, SMI led to a reduction of postprandial influx of free amino acids, hypoactivated target of rapamycin signalling and a hyperactivated amino acid response pathway after refeeding, a status associated with reduced protein synthesis, impaired postprandial glycolysis and lipogenesis. These differential effects were not ameliorated by matching an EAA profile of soyabean meal to that of the FM diet through dietary amino acid supplementation. Therefore, this study demonstrated that the FM diet and SMI diets led to distinct nutrient-sensing responses, which in turn modulated metabolism and determined the utilisation efficiency of diets. Our results provide a new molecular explanation for the role of nutrient sensing in the inferior performance of aquafeeds in which FM is replaced by soyabean meal.

A total of forty weaned pigs ((Landrace×Yorkshire)×Duroc) were used to evaluate the effects of Lactobacillus acidophilus on inflammatory activity after lipopolysaccharide (LPS) challenge. Experimental treatments were as follows: (T1) control diet+saline challenge; (T2) control diet with 0·1 % L. acidophilus+saline challenge; (T3) control diet+LPS challenge; and (T4) control diet with 0·1 % L. acidophilus+LPS challenge. On d-14, piglets were challenged with saline (T1 and T2) or LPS (T3 and T4). Blood samples were obtained at 0, 2, 4, 6 and 12 h after being challenged and analysed for immune cell cytokine production and gene expression pattern. The L. acidophilus treatment increased the average daily weight gain (ADWG) and average daily feed intake (ADFI) compared with the control diet. With the control diet, the LPS challenge (T3) increased the number of immune cells and expression of TNF-α and IL-6 compared with the saline challenge (T1). Whereas with the saline challenge L. acidophilus treatment (T2) increased the number of leucocytes and CD4 compared with the control diet (T1), with the LPS challenge L. acidophilus treatment (T4) decreased the number of leucocytes, lymphocytes, CD4+ and CD8+ and expression of TNF-α and IL-6 compared with the control diet (T3). L. acidophilus treatment decreased the expression of TRL4 and NF-κB in peripheral blood mononuclear cells (PBMC) after LPS challenge, which leads to inhibition of TNF-α, IFN-γ, IL-6, IL-8 and IL1B1 and to induction of IL-4 and IL-10. We suggested that L. acidophilus improved ADWG and ADFI and protected against LPS-induced inflammatory responses by regulating TLR4 and NF-κB expression in porcine PBMC.

Peripheral blood mononuclear cells (PBMC) are increasingly used for nutrigenomic studies. In this study, we aimed to identify whether these cells could reflect the development of an obesogenic profile associated with the intake of high-fat (HF) diets. We analysed, by real-time RT-PCR, the dietary response of key genes related to lipid metabolism, obesity and inflammation in PBMC of control rats, rats fed a cafeteria or a commercial HF diet and rats fed a control diet after the intake of a cafeteria diet (post-cafeteria model). Cafeteria diet intake, which resulted in important overweight and related complications, altered the expressions of most of the studied genes in PBMC, evidencing the development of an obesogenic profile. Commercial HF diet, which produced metabolic alterations but in the absence of noticeably increased body weight, also altered PBMC gene expression, inducing a similar regulatory pattern as that observed for the cafeteria diet. Regulation of carnitine palmitoyltransferase I (Cpt1a) mRNA expression was of special interest; its expression reflected metabolic alterations related to the intake of both obesogenic diets (independently of increased body weight) even at an early stage as well as metabolic recovery in post-cafeteria animals. Thus, PBMC constitute an important source of biomarkers that reflect the increased adiposity and metabolic deregulation associated with the intake of HF diets. In particular, we propose an analysis of Cpt1a expression as a good biomarker to detect the early metabolic alterations caused by the consumption of hyperlipidic diets, and also as a marker of metabolic recovery associated to weight loss.

The whole-body transcriptome of trout alevins was characterised to investigate the effects of long-term feeding of rainbow trout broodstock females a diet free of fishmeal and fish oil on the metabolic capacities of progeny. Effects were studied before first feeding and after 3 weeks of feeding diets containing different proportions of marine and plant ingredients. Feeding alevins plant-based diets resulted in lower fish body weight, irrespective of maternal nutritional history. No differences in whole-body lipids were found between treatments, and the tissue fatty acid profile strongly reflected that of the respective broodstock or first-feeding diets. We showed that the maternal diet history did not significantly affect expressions of any genes before the first feeding. Interestingly, we found an effect of maternal nutritional history on gene expression in alevins after 3 weeks of feeding. The major differences in the transcriptome of alevins from plant-based diet-fed females compared with those from commercial-fed females were as follows: (i) down-regulation of genes involved in muscle growth/contraction and (ii) up-regulation of genes involved in carbohydrate and energy metabolism related to the delay in growth/development observed with plant-based diets. Our findings also showed an effect of the first-feeding diets, irrespective of maternal nutritional history. Specifically, the introduction of plant ingredients resulted in the up-regulation of genes involved in amino acid/protein and cholesterol metabolism and in differences in the expressions of genes related to carbohydrate metabolism. Information gained through this study opens up avenues for further reduction of marine ingredients in trout diets, including the whole rearing cycle.

Gestational diabetes mellitus (GDM) is a serious problem growing worldwide that needs to be addressed with urgency in consideration of the resulting severe complications for both mother and fetus. Growing evidence indicates that a healthy diet rich in fruit, vegetables, nuts, extra-virgin olive oil and fish has beneficial effects in both the prevention and management of several human diseases and metabolic disorders. In this review, we discuss the latest data concerning the effects of dietary bioactive compounds such as polyphenols and PUFA on the molecular mechanisms regulating glucose homoeostasis. Several studies, mostly based on in vitro and animal models, indicate that dietary polyphenols, mainly flavonoids, positively modulate the insulin signalling pathway by attenuating hyperglycaemia and insulin resistance, reducing inflammatory adipokines, and modifying microRNA (miRNA) profiles. Very few data about the influence of dietary exposure on GDM outcomes are available, although this approach deserves careful consideration. Further investigation, which includes exploring the ‘omics’ world, is needed to better understand the complex interaction between dietary compounds and GDM.

Diets rich in fruits and vegetables (FV), which contain (poly)phenols, protect against age-related inflammation and chronic diseases. T-lymphocytes contribute to systemic cytokine production and are modulated by FV intake. Little is known about the relative potency of different (poly)phenols in modulating cytokine release by lymphocytes. We compared thirty-one (poly)phenols and six (poly)phenol mixtures for effects on pro-inflammatory cytokine release by Jurkat T-lymphocytes. Test compounds were incubated with Jurkat cells for 48 h at 1 and 30 µm, with or without phorbol ester treatment at 24 h to induce cytokine release. Three test compounds that reduced cytokine release were further incubated with primary lymphocytes at 0·2 and 1 µm for 24 h, with lipopolysaccharide added at 5 h. Cytokine release was measured, and generation of H2O2 by test compounds was determined to assess any potential correlations with cytokine release. A number of (poly)phenols significantly altered cytokine release from Jurkat cells (P<0·05), but H2O2 generation did not correlate with cytokine release. Resveratrol, isorhamnetin, curcumin, vanillic acid and specific (poly)phenol mixtures reduced pro-inflammatory cytokine release from T-lymphocytes, and there was evidence for interaction between (poly)phenols to further modulate cytokine release. The release of interferon-γ induced protein 10 by primary lymphocytes was significantly reduced following treatment with 1 µm isorhamnetin (P<0·05). These results suggest that (poly)phenols derived from onions, turmeric, red grapes, green tea and açai berries may help reduce the release of pro-inflammatory mediators in people at risk of chronic inflammation.

The isolated or combined effects of betaine and arginine supplementation of reduced protein diets (RPD) on fat content, fatty acid composition and mRNA levels of genes controlling lipid metabolism in pig m. longissimus lumborum and subcutaneous adipose tissue (SAT) were assessed. The experiment was performed on forty intact male pigs (Duroc×Large White×Landrace cross-breed) with initial and final live weights of 60 and 93 kg, respectively. Pigs were randomly assigned to one of the following five diets (n 8): 16·0 % of crude protein (control), 13·0 % of crude protein (RPD), RPD supplemented with 0·33 % of betaine, RPD supplemented with 1·5 % of arginine and RPD supplemented with 0·33 % of betaine and 1·5 % of arginine. Data confirmed that RPD increase intramuscular fat (IMF) content and total fat content in SAT. The increased total fat content in SAT was accompanied by higher GLUT type 4, lipoprotein lipase and stearoyl-CoA desaturase mRNA expression levels. In addition, the supplementation of RPD with betaine and/or arginine did not affect either IMF or total fat in SAT. However, dietary betaine supplementation slightly affected fatty acid composition in both muscle and SAT. This effect was associated with an increase of carnitine O-acetyltransferase mRNA levels in SAT but not in muscle, which suggests that betaine might be involved in the differential regulation of some key genes of lipid metabolism in pig muscle and SAT. Although the arginine-supplemented diet decreased the mRNA expression level of PPARG in muscle and SAT, it did not influence fat content or fatty acid composition in any of these pig tissues.

Combined in vivo (ileal cannulated pig) and in vitro (faecal inoculum-based fermentation) digestion methodologies were used to predict the production and absorption of SCFA in the hindgut of growing pigs. Ileal and faecal samples were collected from animals (n 7) fed diets containing either 25 or 50 g/kg DM of kiwifruit fibre from added kiwifruit for 14 d. Ileal and faecal SCFA concentrations normalised for food DM intake (DMI) and nutrient digestibility were determined. Ileal digesta were collected and fermented for 38 h using a fresh pig faecal inoculum to predict SCFA production. The predicted hindgut SCFA production along with the determined ileal and faecal SCFA were then used to predict SCFA absorption in the hindgut and total tract organic matter digestibility. The determined ileal and faecal SCFA concentrations (e.g. 8·5 and 4·4 mmol/kg DMI, respectively, for acetic acid for the low-fibre diet) represented only 0·2–3·2 % of the predicted hindgut SCFA production (e.g. 270 mmol/kg DMI for acetic acid). Predicted production and absorption of acetic, butyric and propionic acids were the highest for the high-fibre diet (P<0·05), but these inter-diet differences were not observed for the ileal and faecal SCFA concentrations (P>0·05). In conclusion, determined ileal and faecal SCFA concentrations represent only a small fraction of total SCFA production, and may therefore be misleading in relation to the effect of diets on SCFA production and absorption. Considerable quantities of SCFA are produced and absorbed in the hindgut of the pig by the fermentation of kiwifruit.

A total of twenty-four Yorkshire gilt pigs of 6–7 weeks of age were used in a 2×2 factorial experiment to determine the individual and combined effects of the inclusion of two dietary factors (cholesterol rich, 3 % β-cyclodextrin (BCD) and Lactobacillus acidophilus cultures) on total cholesterol and LDL-cholesterol levels in blood serum. Pigs were assigned randomly to treatment groups (n 6). Total serum cholesterol concentrations decreased after 3 weeks in all the experimental treatment groups, including diets with BCD, L. acidophilus or both. Similar trends were observed for serum LDL-cholesterol concentrations among the experimental treatments. No statistically significant differences from the control group were observed in either total serum cholesterol or LDL-cholesterol concentrations (P<0·05) for each of the individual treatment groups: BCD or L. acidophilus. However, significant differences in total serum cholesterol concentrations were observed when comparing the combined treatment group (BCD and L. acidophilus) with the control group, which consisted of a basal diet and sterile milk. The combined treatment group exhibited 17·9 % lower total serum cholesterol concentration after 3 weeks. Similar significant differences were observed when comparing the combined effect experimental group with the control group after 3 weeks. The combined treatment group exhibited 27·9 % lower serum LDL-cholesterol concentrations.

Dietary cholesterol induces hepatic inflammation and fibrosis in animals. We aimed to determine whether dietary cholesterol affects liver-related mortality in hepatitis C virus (HCV)-infected patients. We performed a retrospective cohort study using extended follow-up data from the Hepatitis C Antiviral Long-Term Treatment Against Cirrhosis Trial. The study included HCV patients with advanced fibrosis and compensated cirrhosis. The analysis included 657 patients who completed two FFQ. We assessed whether cholesterol intake, measured in mg/4184 kJ (mg/1000 kcal) of energy intake, was associated with liver-related death or transplantation. In 4·7 (sd 1·6) years, the incidence of liver-related death (n 46) or transplantation (n 52) was 31·8/1000 person-years. The relationship between cholesterol intake and liver-related death or transplantation was significantly different between men and women (test for interaction, P value=0·01). Each higher quartile of cholesterol intake was associated with an increased risk for liver-related death or transplantation in women (adjusted hazard ratio (AHR) 1·83; 95 % CI 1·12, 2·99; Ptrend=0·02), but not in men (AHR 0·96; 95 % CI 0·76, 1·22; Ptrend=0·73). Compared with women whose cholesterol intake was within the recommended guidelines (300 mg/d with a 8368 kJ (2000 kcal) diet), women who consumed more cholesterol had significantly increased risk for liver-related death or transplantation (AHR 4·04; 95 % CI 1·42, 11·5). High dietary cholesterol was associated with an increased risk for liver-related death and transplantation in HCV-infected women with advanced fibrosis or compensated cirrhosis. Future studies should assess whether reducing cholesterol intake, among women who consume an excessive amount, can decrease HCV-related mortality.

A sufficient but balanced vitamin supplementation is a prerequisite for a satisfactory growth pattern and an effective immune system in mink and all other species. The fat-soluble vitamins are very sensitive to over- or under-supply because they interact with each other with respect to dose–response and chemical form. The purpose of the present study was to investigate the effect of increasing the amount of retinol in combination with RRR-α-tocopherol or all-rac-α-tocopherol in the feed given to growing mink on their retinol, cholecalciferol and α-tocopherol concentrations in plasma and selected organs. The results showed that the mink met their retinol requirements from the basal diet, but there were no negative effects of supplying various amounts of retinol on their plasma α-tocopherol concentrations. On the other hand, the study showed that the cholecalciferol status in plasma, assessed as the 25-hydroxycholecalciferol concentration, was low when retinol was supplemented in the feed at high levels. In addition, supplementation with RRR-α-tocopherol in the feed negatively affected the plasma concentration of 25-hydroxycholecalciferol compared with supplementation with all-rac-α-tocopherol. In general, female mink had higher concentrations of fat-soluble vitamins in plasma than male mink.

The aim of the present study was to assess the effects of dietary supplementation with epidermal growth factor (EGF)-expressing Saccharomyces cerevisiae on duodenal development in weaned piglets. In total, forty piglets weaned at 21–26 d of age were assigned to one of the five groups that were provided basic diet (control group) or diet supplemented with S. cerevisiae expressing either empty-vector (INVSc1(EV) group), tagged EGF (T-EGF) (INVSc1-TE(−) group), extracellular EGF (EE-EGF) (INVSc1-EE(+) group) or intracellular EGF (IE-EGF) (INVSc1-IE(+) group). All treatments were delivered as 60·00 μg/kg body weight EGF/d. On 0, 7, 14 and 21 d, eight piglets per treatment were sacrificed to analyse the morphology, activities and mRNA expressions of digestive enzymes, as well as Ig levels (IgA, IgM, IgG) in duodenal mucosa. The results showed significant improvement on 7, 14 and 21 d, with respect to average daily gain (P<0·05), mucosa morphology (villus height and crypt depth) (P<0·05), Ig levels (P<0·01), activities and mRNA expressions of digestive enzymes (creatine kinase, alkaline phosphatase, lactate dehydrogenase and sucrase) (P<0·05) and the mRNA expression of EGF-receptor (P<0·01) in NVSc1-TE(−), INVSc1-EE(+) and INVSc1-IE(+) groups compared with control and INVSc1(EV) groups. In addition, a trend was observed in which the INVSc1-IE(+) group showed an improvement in Ig levels (0·05<P<0·10), mRNA expressions of digestive enzymes and EGF-receptor (P<0·05) compared with NVSc1-TE(−) and INVSc1-EE(+) groups. These results indicate that supplementing recombinant EGF-expressing S. cerevisiae to the diet of weaned piglets enhanced duodenal development. Moreover, biological activity (Ig levels, mRNA expressions of digestive enzymes and EGF-receptor) of IE-EGF was better than either EE-EGF or T-EGF.

Some effects of parasitism, endotoxaemia or sepsis can be mitigated by provision of extra protein. Supplemented protein may encompass a metabolic requirement for specific amino acids (AA). The current study investigates a method to identify and quantify the amounts of AA required during inflammation induced by an endotoxin challenge. One of each pair of six twin sheep was infused in the jugular vein for 20 h with either saline (control) or lipopolysaccharide (LPS, 2 ng/kg body weight per min) from Escherichia coli. Between 12 and 20 h a mixture of stable isotope-labelled AA was infused to measure irreversible loss rates. From 16 to 20 h all sheep were supplemented with a mixture of unlabelled AA infused intravenously. Blood samples were taken before the start of infusions, and then continuously over intervals between 14 and 20 h. At 20 h the sheep were euthanised, and liver and kidney samples were taken for measurement of serine-threonine dehydratase (SDH) activity. LPS infusion decreased plasma concentrations of most AA (P<0·05; P<0·10 for leucine and tryptophan), except for phenylalanine (which increased P=0·022) and tyrosine. On the basis of the incremental response to the supplemental AA, arginine, aspartate, cysteine, glutamate, lysine (tendency only), glycine, methionine, proline, serine and threonine were important in the metabolic response to the endotoxaemia. The AA infusion between 16 and 20 h restored the plasma concentrations in the LPS-treated sheep for the majority of AA, except for glutamine, isoleucine, methionine, serine and valine. LPS treatment increased (P<0·02) SDH activity in both liver and kidney. The approach allows quantification of key AA required during challenge situations.

During pregnancy, glycine and serine become more important because they are the primary suppliers of methyl groups for the synthesis of fetal DNA, and more glycine is required for fetal collagen synthesis as pregnancy progresses. In an earlier study, we reported that glycine flux decreased by 39 % from the first to the third trimester in pregnant adolescent girls. As serine is a primary precursor for glycine synthesis, the objective of this study was to measure and compare glycine and serine fluxes and inter-conversions in pregnant adolescent girls and adult women in the first and third trimesters. Measurements were made after an overnight fast by continuous intravenous infusions of 2H2-glycine and 15N-serine in eleven adolescent girls (17·4 (se 0·1) years of age) and in ten adult women (25·8 (se 0·5) years of age) for 4 h. Adolescent girls had significantly slower glycine flux and they made less glycine from serine in the third (P<0·05) than in the first trimester. Baby birth length was significantly shorter of adolescent girls (P=0·04) and was significantly associated with third trimester glycine flux. These findings suggest that the pregnant adolescent cannot maintain glycine flux in late pregnancy compared with early pregnancy because of decreased synthesis from serine. It is possible that the inability to maintain glycine synthesis makes her fetus vulnerable to impaired cartilage synthesis, and thus linear growth.

Increased substitution of marine ingredients by terrestrial plant products in aquafeeds has been proven to be suitable for Atlantic salmon farming. However, a reduction in n-3 long-chain PUFA is a consequence of this substitution. In contrast, relatively little attention has been paid to the effects of fishmeal and oil substitution on levels of micronutrients such as Se, considering fish are major sources of this mineral for human consumers. To evaluate the effects of dietary marine ingredient substitution on tissue Se distribution and the expression of Se metabolism and antioxidant enzyme genes, Atlantic salmons were fed three feeds based on commercial formulations with increasing levels of plant proteins (PP) and vegetable oil. Lipid content in flesh did not vary at any sampling point, but it was higher in the liver of 1 kg of fish fed higher PP. Fatty acid content reflected dietary input and was related to oxidation levels (thiobarbituric acid-reactive substances). Liver had the highest Se levels, followed by head kidney, whereas the lowest contents were found in brain and gill. The Se concentration of flesh decreased considerably with high levels of substitution, reducing the added value of fish consumption. Only the brain showed significant differences in glutathione peroxidase, transfer RNA selenocysteine 1-associated protein 1b and superoxide dismutase expression, whereas no significant regulation of Se-related genes was found in liver. Although Se levels in the diets satisfied the essential requirements of salmon, high PP levels led to a reduction in the supply of this essential micronutrient.

Type 2 diabetes (T2D) is a major risk factor of CVD. The effects of purified sardine proteins (SP) were examined on glycaemia, insulin sensitivity and reverse cholesterol transport in T2D rats. Rats fed a high-fat diet (HFD) for 5 weeks, and injected with a low dose of streptozotocin, were used. The diabetic rats were divided into four groups, and they were fed casein (CAS) or SP combined with 30 or 5 % lipids, for 4 weeks. HFD-induced hyperglycaemia, insulin resistance and hyperlipidaemia in rats fed HFD, regardless of the consumed protein. In contrast, these parameters lowered in rats fed SP combined with 5 or 30 % lipids, and serum insulin values reduced in SP v. CAS. HFD significantly increased total cholesterol and TAG concentrations in the liver and serum, whereas these parameters decreased with SP, regardless of lipid intake. Faecal cholesterol excretion was higher with SP v. CAS, combined with 30 or 5 % lipids. Lecithin:cholesterol acyltransferase (LCAT) activity and HDL3-phospholipids (PL) were higher in CAS-HF than in CAS, whereas HDL2-cholesteryl esters (CE) were lower. Otherwise, LCAT activity and HDL2-CE were higher in the SP group than in the CAS group, whereas HDL3-PL and HDL3-unesterified cholesterol were lower. Moreover, LCAT activity lowered in the SP-HF group than in the CAS-HF group, when HDL2-CE was higher. In conclusion, these results indicate the potential effects of SP to improve glycaemia, insulin sensitivity and reverse cholesterol transport, in T2D rats.

Plant lignans are diphenolic compounds ingested with whole grains and seeds and converted to enterolignans by the colonic microbiota. In the present study, we investigated absorption and metabolism of plant lignans and enterolignans in vivo after consumption of cereal-based diets. Six pigs fitted with catheters in the mesenteric artery and portal vein and with a flow probe attached to the portal vein along with twenty pigs for quantitative collection of urine were used for this study. The animals were fed bread based on wheat flour low in plant lignans and three lignan-rich breads based on whole-wheat grain, wheat aleurone flour or rye aleurone flour. Plant lignans and enterolignans in plasma were monitored daily at fast after 0–3 d of lignan-rich intake, and on the 4th day of lignan-rich intake a 10-h profile was completed. Urine samples were collected after 11 d of lignan-rich diet consumption. The concentrations of plant lignans were low at fast, and was 1·2–2·6 nmol/l after switching from the low-lignan diet to the lignan-rich diets. However, on the profile day, the concentration and quantitative absorption of plant lignans increased significantly from 33 nmol/h at fast to 310 nmol/h 0–2·5 h after ingestion with a gradual increase in the following periods. Quantitatively, the absorption of plant lignans across diets amounted to 7 % of ingested plant lignans, whereas the urinary excretion of plant lignans was 3 % across diets. In conclusion, there is a substantial postprandial uptake of plant lignans from cereals, suggesting that plant lignans are absorbed from the small intestine.

Four isolipidic and isoenergetic diets with different protein:carbohydrate (CH) contents (48:38, 52:34, 56:30, 60:26) were fed to juvenile Senegalese sole (22·01 (sem 0·01) g) during 104 d. Oral glucose tolerance tests were performed at the beginning (4 d) and at the end (104 d) of the experiment to assess the effect of the dietary treatment on glucose tolerance. Samples of blood, liver and muscle of all dietary groups were also obtained at the initial and final phases of the trial at different postprandial times (0, 1, 5 and 10 h after feeding) in order to analyse glucose and NEFA in plasma, and metabolites and enzyme activities involved in glycogen metabolism, glycolysis, gluconeogenesis and lipogenesis pathways in liver and muscle. The results obtained in this study suggest a good glucose tolerance in Senegalese sole. This species tolerated important amounts of CH in the diet without showing any deleterious signs in terms of growth or any metabolic disorders. After 104 d of feeding diets with an important amount of CH (48:38 and 52:34), the control of glycaemia was maintained and even postprandial glucose levels in plasma were (in general) lower than at the beginning of the experiment. This reasonable tolerance to glucose is also reflected by an increased use of glucose through glycolysis in liver (indicated by glucokinase activity), and the absence of changes in lipogenic potential in the same tissue (indicated by ATP citrate lyase activity). No clear changes were induced in the muscle by the dietary treatments.

The intermittent energy restriction (IER) approach to weight loss involves short periods of substantial (75–100 %) energy restriction (ER) interspersed with normal eating. This study aimed to characterise the early metabolic response to these varying degrees of ER, which occurs acutely and prior to weight loss. Ten (three female) healthy, overweight/obese participants (36 (SEM 5) years; 29·0 (sem 1·1) kg/m2) took part in this acute three-way cross-over study. Participants completed three 1-d dietary interventions in a randomised order with a 1-week washout period: isoenergetic intake, partial 75 % ER and total 100 % ER. Fasting and postprandial (6-h) metabolic responses to a liquid test meal were assessed the following morning via serial blood sampling and indirect calorimetry. Food intake was also recorded for two subsequent days of ad libitum intake. Relative to the isoenergetic control, postprandial glucose responses were increased following total ER (+142 %; P=0·015) and to a lesser extent after partial ER (+76 %; P=0·051). There was also a delay in the glucose time to peak after total ER only (P=0·024). Both total and partial ER interventions produced comparable reductions in postprandial TAG responses (−75 and −59 %, respectively; both P<0·05) and 3-d energy intake deficits of approximately 30 % (both P=0·015). Resting and meal-induced thermogenesis were not significantly affected by either ER intervention. In conclusion, our data demonstrate the ability of substantial ER to acutely alter postprandial glucose–lipid metabolism (with partial ER producing the more favourable overall response), as well as incomplete energy-intake compensation amongst overweight/obese participants. Further investigations are required to establish how metabolism adapts over time to the repeated perturbations experienced during IER, as well as the implications for long-term health.

An efficient and robust method to measure vitamin D (25-hydroxy vitamin D3 (25(OH)D3) and 25-hydroxy vitamin D2 in dried blood spots (DBS) has been developed and applied in the pan-European multi-centre, internet-based, personalised nutrition intervention study Food4Me. The method includes calibration with blood containing endogenous 25(OH)D3, spotted as DBS and corrected for haematocrit content. The methodology was validated following international standards. The performance characteristics did not reach those of the current gold standard liquid chromatography-MS/MS in plasma for all parameters, but were found to be very suitable for status-level determination under field conditions. DBS sample quality was very high, and 3778 measurements of 25(OH)D3 were obtained from 1465 participants. The study centre and the season within the study centre were very good predictors of 25(OH)D3 levels (P<0·001 for each case). Seasonal effects were modelled by fitting a sine function with a minimum 25(OH)D3 level on 20 January and a maximum on 21 July. The seasonal amplitude varied from centre to centre. The largest difference between winter and summer levels was found in Germany and the smallest in Poland. The model was cross-validated to determine the consistency of the predictions and the performance of the DBS method. The Pearson’s correlation between the measured values and the predicted values was r 0·65, and the sd of their differences was 21·2 nmol/l. This includes the analytical variation and the biological variation within subjects. Overall, DBS obtained by unsupervised sampling of the participants at home was a viable methodology for obtaining vitamin D status information in a large nutritional study.