Low doses of diarrhoeagenic E. coli induce enhanced monocyte and mDC responses and prevent development of symptoms after homologous rechallenge.

The experimental challenge with attenuated enterotoxigenic E. coli strain E1392/75-2A prevents diarrhea upon a secondary challenge with the same bacteria. A dose-response pilot study was performed to investigate which immunological factors are associated with this protection. Healthy subjects were inoculated with increasing E. coli doses of 1E6-1E10 CFU, and three weeks later, all participants were rechallenged with the highest dose (1E10 CFU). Gastrointestinal discomfort symptoms were recorded, and stool and blood samples were analyzed. After the primary challenge, stool frequency, diarrhea symptom scores, and E. coli-specific serum IgG (IgG-CFA/II) titer increased in a dose-dependent manner. Fecal calprotectin and serum IgG-CFA/II response after primary challenge were delayed in the lower dose groups. Even though stool frequency after the secondary challenge was inversely related to the primary inoculation dose, all E. coli doses protected against clinical symptoms upon rechallenge. Ex vivo stimulation of PBMCs with E. coli just before the second challenge resulted in increased numbers of IL-6+/TNF-α+ monocytes and mDCs than before the primary challenge, without dose-dependency. These data demonstrate that primary E. coli infection with as few as 1E6 CFU protects against a high-dose secondary challenge with a homologous attenuated strain. Increased serum IgG-CFA/II levels and E. coli-induced mDC and monocyte responses after primary challenge suggest that protection against secondary E. coli challenges is associated with adaptive as well as innate immune responses.

Effects of Dietary Supplementation with Carrot-Derived Rhamnogalacturonan-I (cRG-I) on Accelerated Protective Immune Responses and Quality of Life in Healthy Volunteers Challenged with Rhinovirus in a Randomized Trial.

An adequate and balanced supply of nutrients is essential for maintaining health, and an optimal immune response is fast, contained and properly controlled, curbing infections quickly while minimizing damage. Several micronutrients contribute to normal immune function and certain dietary fibers, for example pectic polysaccharides, can play an important role in educating and regulating immune cell responses. The aim of this paper is to elaborate on our initial findings that dietary supplementation with carrot-derived rhamnogalacturonan-I (cRG-I) accelerates and augments local innate immune and anti-viral interferon response to a rhinovirus-16 (RV16) infection and reduces the severity and duration of symptoms in humans. Dietary intake of cRG-I also enhanced immune responses to this respiratory viral infection as measured by ex vivo stimulation of whole blood with the Toll-like receptor 3 (TLR3) ligand polyinosinic:polycytidylic acid and NK cell function. Consumption of cRG-I also reduced the negative effects of this common cold infection on quality of life as assessed by individual symptom scores. RG-I from carrot is a safe, sustainable, and economically viable solution that could easily be integrated into food products and dietary supplements aiming to support immune fitness and wellbeing.

The Dietary Intake of Carrot-Derived Rhamnogalacturonan-I Accelerates and Augments the Innate Immune and Anti-Viral Interferon Response to Rhinovirus Infection and Reduces Duration and Severity of Symptoms in Humans in a Randomized Trial.

Acute respiratory infections are an important health concern. Traditionally, polysaccharide-enriched extracts from plants, containing immunomodulatory rhamnogalacturonan-I (RG-1), were used prophylactically. We established the effects of dietary supplementation with carrot-derived RG-I (cRG-I, 0-0.3-1.5 g/day) in 177 healthy individuals (18-65 years) on symptoms following infection with rhinovirus strain 16 (RV16). Primary outcomes were changes in severity and duration of symptoms, and viral load in nasal lavage. Secondary outcomes were changes in innate immune and anti-viral responses, reflected by CXCL10 and CXCL8 levels and cell differentials in nasal lavage. In a nested cohort, exploratory transcriptome analysis was conducted on nasal epithelium. Intake of cRG-I was safe, well-tolerated and accelerated local cellular and humoral innate immune responses induced by RV16 infection, with the strongest effects at 1.5 g/d. At 0.3 g/d, a faster interferon-induced response, induction of the key anti-viral gene EIF2AK2, faster viral clearance, and reduced symptom severity (-20%) and duration (-25%) were observed. Anti-viral responses, viral clearance and symptom scores at 1.5 g/d were in between those of 0 and 0.3 g/d, suggesting a negative feedback loop preventing excessive interferon responses. Dietary intake of cRG-I accelerated innate immune and antiviral responses, and reduced symptoms of an acute respiratory viral infection.

Protein supplementation during an energy-restricted diet induces visceral fat loss and gut microbiota amino acid metabolism activation: a randomized trial.

Interactions between diet and gut microbiota are critical regulators of energy metabolism. The effects of fibre intake have been deeply studied but little is known about the impact of proteins. Here, we investigated the effects of high protein supplementation (Investigational Product, IP) in a double blind, randomised placebo-controled intervention study (NCT01755104) where 107 participants received the IP or an isocaloric normoproteic comparator (CP) alongside a mild caloric restriction. Gut microbiota profiles were explored in a patient subset (n = 53) using shotgun metagenomic sequencing. Visceral fat decreased in both groups (IP group: - 20.8 ± 23.2 cm2; CP group: - 14.5 ± 24.3 cm2) with a greater reduction (p < 0.05) with the IP supplementation in the Per Protocol population. Microbial diversity increased in individuals with a baseline low gene count (p < 0.05). The decrease in weight, fat mass and visceral fat mass significantly correlated with the increase in microbial diversity (p < 0.05). Protein supplementation had little effects on bacteria composition but major differences were seen at functional level. Protein supplementation stimulated bacterial amino acid metabolism (90% amino-acid synthesis functions enriched with IP versus 13% in CP group (p < 0.01)). Protein supplementation alongside a mild energy restriction induces visceral fat mass loss and an activation of gut microbiota amino-acid metabolism.Clinical trial registration: NCT01755104 (24/12/2012). https://clinicaltrials.gov/ct2/show/record/NCT01755104?term=NCT01755104&draw=2&rank=1 .

Vitamin D Serum Levels in the UK Population, including a Mathematical Approach to Evaluate the Impact of Vitamin D Fortified Ready-to-Eat Breakfast Cereals: Application of the NDNS Database.

Vitamin D status is relatively poor in the general population, potentially leading to various conditions. The present study evaluates the relationship between vitamin D status and intake in the UK population and the impact of vitamin D fortified ready-to-eat cereals (RTEC) on this status via data from the National Diet and Nutrition Survey (NDNS: 2008-2012). Four cohorts were addressed: ages 4-10 (n = 803), ages 11-18 (n = 884), ages 19-64 (n = 1655) and ages 65 and higher (n = 428). The impact of fortification by 4.2 µg vitamin D per 100 g of RTEC on vitamin D intake and status was mathematically modelled. Average vitamin D daily intake was age-dependent, ranging from ~2.6 (age range 4-18 years) to ~5.0 µg (older than 64 years). Average 25(OH)D concentration ranged from 43 to 51 nmol/L, the highest in children. The relationship between vitamin D intake and status followed an asymptotic curve with a predicted plateau concentration ranging from 52 in children to 83 nmol/L in elderly. The fortification model showed that serum concentrations increased with ~1.0 in children to ~6.5 nmol/L in the elderly. This study revealed that vitamin D intake in the UK population is low with 25(OH)D concentrations being suboptimal for general health. Fortification of breakfast cereals can contribute to improve overall vitamin D status.

Helianthus annuus Seed Extract Affects Weight and Body Composition of Healthy Obese Adults during 12 Weeks of Consumption: A Randomized, Double-Blind, Placebo-Controlled Pilot Study.

The aim of this pilot study was to evaluate the effects of a sunflower (Helianthus annuus) seed extract, standardized for 40% chlorogenic acids on weight and body composition of obese adults. Fifty subjects were randomly assigned to sunflower extract or isocaloric placebo groups, receiving respectively 500 mg/day of treatment for 12 weeks. At the end of the intervention, a significant decrease in body weight, Body Mass Index (BMI), and waist circumference was observed, especially for obese female subjects above 30 years. Those changes were associated with modified body composition related to fat mass loss. A decrease in blood cholesterol was also observed, supporting the potential action of sunflower extract on lipid metabolism. It was concluded that consumption of sunflower extract has a beneficial effect on body weight, fat mass, and lipid profile, providing evidence for its use as a natural anti-obesity herbal extract.

Effects of ß-Fructans Fiber on Bowel Function: A Systematic Review and Meta-Analysis.

The aim of this systematic review and meta-analysis was to assess the effects of ß-fructan supplementation on bowel function in healthy volunteers and patients. The search process was based on the selection of publications listed in the Pubmed and EUPMC database until December 2017, plus two unpublished studies, to identify studies evaluating the impact of ß-fructans on bowel movement and stool parameters. Forty-seven publications were selected for inclusion. Primary parameter was frequency of bowel movements, evaluated by the number of defecations per day during the study period. Secondary outcomes were stool consistency, stool dry and wet weights, and transit time. Short-chain (DP < 10) ß-fructans contributed to increased stool frequency (0.36 defecation +/- 0.06 per day; p < 0.001), while no significant effect was reported with long-chain (DP ≥ 10) ß-fructans (-0.03 +/- 0.11, p = 0.82). A minimal increase in stool wet weight was also statistically demonstrated with short-chain ß-fructans. Moreover, the meta-analysis highlighted significant differences in stool consistency in contrast to fecal dry weight after ß-fructan supplementation. This systematic review and meta-analysis indicates that short-chain ß-fructan supplementation has a positive effect on bowel function by significantly increasing the frequency of bowel movements.

The Diuretic Action of Weak and Strong Alcoholic Beverages in Elderly Men: A Randomized Diet-Controlled Crossover Trial.

With ageing, there is a greater risk of dehydration. This study investigated the diuretic effect of alcoholic beverages varying in alcohol concentration in elderly men. Three alcoholic beverages (beer (AB), wine (AW), and spirits (S)) and their non-alcoholic counterparts (non-alcoholic beer (NAB), non-alcoholic wine (NAW), and water (W)) were tested in a diet-controlled randomized crossover trial. For the alcoholic beverages, alcohol intake equaled a moderate amount of 30 g. An equal volume of beverage was given for the non-alcoholic counterpart. After consumption, the urine output was collected every hour for 4 h and the total 24 h urine output was measured. AW and S resulted in a higher cumulative urine output compared to NAW and W during the first 4 h (effect size: 0.25 mL p < 0.003, effect size: 0.18 mL, p < 0.001, respectively), but not after the 24h urine collection (p > 0.40, p > 0.10). AB and NAB did not differ at any time point (effect size: -0.02 mL p > 0.70). For urine osmolality, and the sodium and potassium concentration, the findings were in line. In conclusion, only moderate amounts of stronger alcoholic beverages, such as wine and spirits, resulted in a short and small diuretic effect in elderly men.

Peptides from the variable region of specific antibodies are shared among lung cancer patients.

Late diagnosis of lung cancer is still the main reason for high mortality rates in lung cancer. Lung cancer is a heterogeneous disease which induces an immune response to different tumor antigens. Several methods for searching autoantibodies have been described that are based on known purified antigen panels. The aim of our study is to find evidence that parts of the antigen-binding-domain of antibodies are shared among lung cancer patients. This was investigated by a novel approach based on sequencing antigen-binding-fragments (Fab) of immunoglobulins using proteomic techniques without the need of previously known antigen panels. From serum of 93 participants of the NELSON trial IgG was isolated and subsequently digested into Fab and Fc. Fab was purified from the digested mixture by SDS-PAGE. The Fab containing gel-bands were excised, tryptic digested and measured on a nano-LC-Orbitrap-Mass-spectrometry system. Multivariate analysis of the mass spectrometry data by linear canonical discriminant analysis combined with stepwise logistic regression resulted in a 12-antibody-peptide model which was able to distinguish lung cancer patients from controls in a high risk population with a sensitivity of 84% and specificity of 90%. With our Fab-purification combined Orbitrap-mass-spectrometry approach, we found peptides from the variable-parts of antibodies which are shared among lung cancer patients.

Design of the South East Asian Nutrition Survey (SEANUTS): a four-country multistage cluster design study.

Nutrition is a well-known factor in the growth, health and development of children. It is also acknowledged that worldwide many people have dietary imbalances resulting in over- or undernutrition. In 2009, the multinational food company FrieslandCampina initiated the South East Asian Nutrition Survey (SEANUTS), a combination of surveys carried out in Indonesia, Malaysia, Thailand and Vietnam, to get a better insight into these imbalances. The present study describes the general study design and methodology, as well as some problems and pitfalls encountered. In each of these countries, participants in the age range of 0·5-12 years were recruited according to a multistage cluster randomised or stratified random sampling methodology. Field teams took care of recruitment and data collection. For the health status of children, growth and body composition, physical activity, bone density, and development and cognition were measured. For nutrition, food intake and food habits were assessed by questionnaires, whereas in subpopulations blood and urine samples were collected to measure the biochemical status parameters of Fe, vitamins A and D, and DHA. In Thailand, the researchers additionally studied the lipid profile in blood, whereas in Indonesia iodine excretion in urine was analysed. Biochemical data were analysed in certified laboratories. Study protocols and methodology were aligned where practically possible. In December 2011, data collection was finalised. In total, 16,744 children participated in the present study. Information that will be very relevant for formulating nutritional health policies, as well as for designing innovative food and nutrition research and development programmes, has become available.

Collagen peptides in urine: a new promising biomarker for the detection of colorectal liver metastases.

INTRODUCTION: For both patients and the outpatient clinic the frequent follow-up visits after a resection of colorectal cancer (CRC) are time consuming and due to large patient numbers expensive. Therefore it is important to develop an effective non-invasive test for the detection of colorectal liver metastasis (CRLM) which could be used outside the hospital. The urine proteome is known to provide detailed information for monitoring changes in the physiology of humans. Urine collection is non-invasive and urine naturally occurring peptides (NOPs) have the advantage of being easily accessible without labour-intensive sample preparation. These advantages make it potentially useful for a quick and reliable application in clinical settings. In this study, we will focus on the identification and validation of urine NOPs to discriminate patients with CRLM from healthy controls. MATERIALS AND METHODS: Urine samples were collected from 24 patients with CRLM and 25 healthy controls. In the first part of the study, samples were measured with a nano liquid chromatography (LC) system (Thermo Fisher Scientific, Germaring, Germany) coupled on-line to a hybrid linear ion trap/Orbitrap mass spectrometer (LTQ-Orbitrap-XL, Thermo Fisher Scientific, Bremen, Germany). A discovery set was used to construct the model and consecutively the validation set, being independent from the discovery set, to check the acquired model. From the peptides which were selected, multiple reaction monitoring (MRM's) were developed on a UPLC-MS/MS system. RESULTS: Seven peptides were selected and applied in a discriminant analysis a sensitivity of 84.6% and a specificity of 92.3% were established (Canonical correlation:0.797, Eigenvalue:1.744, F:4.49, p:0.005). The peptides AGPP(-OH)GEAGKP(-OH)GEQGVP(-OH)GDLGA P(-OH)GP and KGNSGEP(-OH)GAPGSKGDTGAKGEP(-OH)GPVG were selected for further quantitative analysis which showed a sensitivity of 88% and a specificity of 88%. CONCLUSION: Urine proteomic analysis revealed two very promising peptides, both part from collagen type 1, AGPP(-OH)GEAGKP(-OH)GEQGVP(-OH)GDLGAP(-OH)GP and KGNSGEP(-OH)GAPGSKGDTGAKGEP(-OH)GPVG which could detect CRLM in a non-invasive manner.

Serum proteomics in amnestic mild cognitive impairment.

We have explored proteins related to mild cognitive impairment (MCI). The serum proteome of 35 amnestic MCI patients and 35 cognitively healthy persons was investigated by LC MS. We identified 108 differentially expressed peptides between MCI patients and controls, belonging to 39 proteins. Eight proteins were selected for further investigation by quantitative protein measurements using a MRM assay; apolipoprotein E, carboxypeptidase N subunit 2, complement factor B (CFAB), galectin-3 binding protein (LG3BP), lumican, serum amyloid A-4 protein (SAA4), serum amyloid P-component, and sex hormone binding globulin. Results of the quantitative protein measurements showed significantly decreased levels of carboxypeptidase N subunit 2, CFAB, LG3BP, SAA4, and serum amyloid P-component in serum from amnestic MCI patients compared with cognitive healthy controls (two-sided t-test; p < 0.05). Apolipoprotein E and lumican showed no significant difference in protein levels, sex hormone binding globulin could not be quantified since the MRM assay did not reach the required sensitivity. A model based on the three most significantly decreased proteins (CFAB, LG3BP, and SAA4) showed a sensitivity and specificity of 73 and 66%, respectively, for the initial sample set. A small external validation set yielded 77% sensitivity and 75% specificity.

The effect of lactose-isomaltulose-containing growing-up milks on cognitive performance of Indonesian children: a cross-over study.

Glycaemic response to dietary carbohydrates might have an impact on cognitive performance. The present study investigated the effects of growing-up milks (GUM) with isomaltulose and extra minerals and vitamins or lower protein content on cognitive parameters in children aged 5­6 years. In a blinded, partly randomised, controlled, cross-over study, four GUM were provided, each taken over 14 d (2 × 200 ml/d): standard (Std) GUM; Std GUM+5 g isomaltulose (Iso-5 GUM); Iso-5 GUM with 26 % less protein (Iso-5 LP GUM); Std GUM with 2·5 g isomaltulose and extra Mg, Zn, Se, D3, B1, B2, B12, folic acid and choline (Iso-2·5 GUM). At test days, when GUM replaced breakfast, repeated (0, 60, 120 and 180 min post-dose) cognitive tasks were performed (picture presentation, simple reaction time, digit vigilance, choice reaction time, spatial and numeric working memory and picture recognition). Task performance of all subjects (n 50) worsened over the morning. Best performance was seen on isomaltulose GUM, most notably at 180 min. Iso-2·5 GUM showed best performance on several parameters of attention and memory, Iso-5 GUM performed best on parameters of memory and Iso-5 LP GUM was positively associated with parameters of attention but less with memory. Std GUM showed only a benefit on one attention and one memory task. Thus, isomaltulose-enriched GUM positively affected parameters of attention and memory at 180 min post-dose when compared with Std GUM. Extra minerals and vitamins seem beneficial, whereas lowering protein content might improve attention in particular.

Dietary galacto-oligosaccharides and calcium: effects on energy intake, fat-pad weight and satiety-related, gastrointestinal hormones in rats.

Galacto-oligosaccharides (GOS) are carbohydrates that are fermented by colonic microbiota. The present study examined effects of a 3-week dietary enrichment with 6 % (w/w) GOS on parameters of energy balance in forty-three male Wistar rats. GOS was tested with two doses of calcium phosphate (30 and 100 mmol/kg), known to differently affect colonic fermentation. After 17 d, isoenergetic test meals were presented and plasma responses of ghrelin, glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) were measured. On day 21 (study termination) epididymal fat pads and caecum were weighed. Additionally, gastrointestinal mucosal samples and proximal colonic contents were analysed for gene expression (ghrelin, proglucagon and PYY) and fermentation metabolites (SCFA and lactate), respectively. GOS reduced energy intake most prominently during the first week, without provoking compensatory overeating later on (average intake reduction: 14 %). The GOS-fed rats showed increased caecal and reduced fat-pad weight and increased gene expression of the satiety-related peptides, PYY (1.7-fold) and proglucagon (3.5-fold). Pre-meal baseline and post-meal plasma levels of PYY, but not of ghrelin or GLP-1, were higher in GOS-fed rats than in control rats. Ca enrichment resulted in higher energy intake (average 4.5 %). GOS diets increased lactic acid levels and slightly reduced butyric acid in proximal colonic contents. Ca abolished the GOS-related elevation of lactic acid, while increasing propionic acid levels, but did not inhibit GOS-related effects on energy intake, fat-pad weight or gene expression. These results indicate that dietary GOS stimulate a number of physiological mechanisms that can reduce energy intake, regardless of the calcium phosphate content of the diet.

Evaluation of satiety enhancement, including compensation, by blends of gum arabic. A methodological approach.

In the present study a potential satiating effect by two blends of gum arabic (EmulGold(®) (EG) and PreVitae(®) (PV)) was investigated in healthy humans applying a regression analysis on the change of values throughout the interval of the study. Two studies were thus conducted: a feasibility study using doses between 10 and 40 g and a dose-finding study of 5 or 10 g of only EG. The gums were dissolved in 250 ml of water (negative control). In both studies energy intake was determined 3 h after consumption, while Visual Analogue Scale (VAS) scores were recorded every 30 min from the time of consumption onwards. At doses of 40 g both EG and PV yielded a significant reduction in energy intake of more than 100 and 200 kcal, respectively. At doses of 10 or 20 g the reduction in energy intake amounted to more than 100 kcal for both. The second study demonstrated a significant reduction in caloric intake of more than 60 kcal at doses of 5 and 10 g of EG. With respect to the subjective perception of satiety, VAS scores revealed a significant increase as compared to the negative control of all doses of both gums. The regression analysis was sensitive in identifying not only the intensity of the perception during the time interval of the study but also the change in this intensity over time. The results of this study show that both blends of gum arabic are able to decrease the caloric intake significantly 3 h after consumption, and increase subjective ratings of feeling satiated, and could therefore be used in a dietary approach to control body weight development.

Description of Acinetobacter venetianus ex Di Cello et al. 1997 sp. nov.

The name 'Acinetobacter venetianus' has been used previously to designate three marine hydrocarbon-degrading Acinetobacter strains, of which strain RAG-1 (=ATCC 31012) has industrial applications for the production of the bioemulsifier emulsan. However, to date, the name of this taxon has not been validly published. In this study, five strains were examined to corroborate the delineation of this taxon by means of phenotypic characterization, DNA-DNA hybridization, selective restriction fragment amplification (AFLP), amplified rDNA restriction analysis (ARDRA), rpoB gene sequence analysis and tRNA intergenic spacer length polymorphism analysis (tDNA-PCR) and to emend the description of 'Acinetobacter venetianus' (ex Di Cello et al. 1997). AFLP analysis showed that the five strains formed a tight cluster at 56.8+/-5.0 % genomic relatedness that was separated from strains of other haemolytic species of the genus Acinetobacter and from the type and reference strains of other Acinetobacter species at

Gum arabic establishes prebiotic functionality in healthy human volunteers in a dose-dependent manner.

The present study was undertaken to determine the prebiotic efficacy of gum arabic upon consumption by man for up to 4 weeks and, if any, to establish the dose-effect relationship. Human healthy volunteers consumed various daily doses (5, 10, 20, 40 g) of gum arabic (EmulGold) in water for up to 4 weeks. Daily consumption of water was taken as the negative control and that of 10 g inulin as the positive control. At 0, 1, 2 and 4 weeks quantification of bacterial numbers in stool samples was performed via real time-PCR techniques and questionnaires were filled in to account for potential drawbacks. The genera of Bifidobacteria and Lactobacilli were taken as potentially beneficial bacteria and those of Bacteroides, Clostridium difficile and Enterococci as potentially non-beneficial, this distinction was dependent on the issue of these numbers being or becoming out of balance in the host. Compared with the negative control the numbers of Bifidobacteria and Lactobacilli 4 weeks after consumption were significantly higher for gum arabic: the optimal dose being around 10 g. Moreover, at this dose the numbers of Bifidobacteria, Lactobacilli and Bacteroides were significantly higher for gum arabic than for inulin. No significant drawback was encountered during the study. It is concluded that gum arabic establishes prebiotic efficacy, at least as good as inulin. The optimal daily dose was found to be 10 g.

The thermogenic and metabolic effects of protein hydrolysate with or without a carbohydrate load in healthy male subjects.

High-protein diets are beneficial in weight maintenance because of their satiating and thermogenic effects. These effects may be partly mediated by the hormonal effects of proteins. This study investigated the effect of soy protein hydrolysate (SPH) with and without a carbohydrate pre- and afterload on energy metabolism and hormonal secretion in 8 healthy nonobese subjects. In an additional trial, pea protein hydrolysate was compared to SPH, both with a carbohydrate afterload. The study had a single-blind crossover design. In all cases, 0.4 g protein and/or carbohydrate per kilogram of body weight was tested. Diet-induced thermogenesis (DIT) was measured by ventilated hood measurements, and postprandial blood samples were drawn over 3 hours. Soy protein hydrolysate consumption induced a higher DIT than a carbohydrate (CHO) load. Both conditions induced similar insulin responses. Soy protein hydrolysate induced a glucagon, but no glucose, response; whereas CHO induced a glucose, but no glucagon, response. Soy protein hydrolysate with a CHO pre- or afterload induced similar DIT and insulin responses. No glucose response was found when SPH preceded the CHO load. Total glucagon responses were similar with CHO as pre- and afterload, but time courses were different. Pea protein hydrolysate with a CHO afterload induced both higher insulin and glucagon responses (area under the curve) than SPH with CHO afterload, but DIT was similar in both conditions. In conclusion, this study shows that the larger DIT after protein than after CHO may be related to the glucagon response that is induced by protein but not by CHO; that the protein-induced DIT and glucagon response are not influenced by a CHO pre- or afterload; and that protein ingestion can fully prevent the plasma glucose increase associated with CHO when CHOs are ingested after proteins.