Risk assessment of nutrients: There must be a threshold for their effects.

Nutrients serve physiological functions in a dose-dependent manner and that needs to be recognized in risk assessment. An example of the consequences of not properly considering this can be seen in a recent assessment by the European Food Safety Authority (EFSA). EFSA concluded in 2022 that the intake of added and free sugars should be "as low as possible in the context of a nutritionally adequate diet". That conclusion of EFSA is based on the effects on two surrogate endpoints for an adverse effect found in randomized controlled trials with high sugars intake levels: fasting glucose and fasting triglycerides. The lowest intake levels in these trials were around 10 energy% and at this intake level there were no adverse effects on the two outcomes. This indicates that the adverse effects of sugars have an observable threshold value for these two endpoints. The most appropriate interpretation from the vast amount of data is that currently no definitive conclusion can be drawn on the tolerable upper intake level for dietary sugars. Therefore, EFSA's own guidance would lead to the conclusion that the available data do not allow the setting of an upper limit for added sugars and hence, that more robust data are required to identify the threshold value for intake of sugars.

Amino acids activate mTORC1 to release roe deer embryos from decelerated proliferation during diapause.

Embryonic diapause in mammals leads to a reversible developmental arrest. While completely halted in many species, European roe deer (Capreolus capreolus) embryos display a continuous deceleration of proliferation. During a 4-mo period, the cell doubling time is 2 to 3 wk. During this period, the preimplantation blastocyst reaches a diameter of 4 mm, after which it resumes a fast developmental pace to subsequently implant. The mechanisms regulating this notable deceleration and reacceleration upon developmental resumption are unclear. We propose that amino acids of maternal origin drive the embryonic developmental pace. A pronounced change in the abundance of uterine fluid mTORC1-activating amino acids coincided with an increase in embryonic mTORC1 activity prior to the resumption of development. Concurrently, genes related to the glycolytic and phosphate pentose pathway, the TCA cycle, and one carbon metabolism were up-regulated. Furthermore, the uterine luminal epithelial transcriptome indicated increased estradiol-17ß signaling, which likely regulates the endometrial secretions adapting to the embryonic needs. While mTORC1 was predicted to be inactive during diapause, the residual embryonic mTORC2 activity may indicate its involvement in maintaining the low yet continuous proliferation rate during diapause. Collectively, we emphasize the role of nutrient signaling in preimplantation embryo development. We propose selective mTORC1 inhibition via uterine catecholestrogens and let-7 as a mechanism regulating slow stem cell cycle progression.

Novel drug transporter substrates identification: An innovative approach based on metabolomic profiling, in silico ligand screening and biological validation.

Solute carrier (SLC) transport proteins are fundamental for the translocation of endogenous compounds and drugs across membranes, thus playing a critical role in disease susceptibility and drug response. Because only a limited number of transporter substrates are currently known, the function of a large number of SLC transporters is elusive. Here, we describe the proof-of-concept of a novel strategy to identify SLC transporter substrates exemplarily for the proton-coupled peptide transporter (PEPT) 2 (SLC15A2) and multidrug and toxin extrusion (MATE) 1 transporter (SLC47A1), which are important renal transporters of drug reabsorption and excretion, respectively. By combining metabolomic profiling of mice with genetically-disrupted transporters, in silico ligand screening and in vitro transport studies for experimental validation, we identified nucleobases and nucleoside-derived anticancer and antiviral agents (flucytosine, cytarabine, gemcitabine, capecitabine) as novel drug substrates of the MATE1 transporter. Our data confirms the successful applicability of this new approach for the identification of transporter substrates in general, which may prove particularly relevant in drug research.

Allulose in human diet: the knowns and the unknowns.

D-Allulose, also referred to as psicose, is a C3-epimer of D-fructose used as a sugar substitute in low energy products. It can be formed naturally during processing of food and drinks containing sucrose and fructose or is prepared by chemical synthesis or via enzymatic treatment with epimerases from fructose. Estimated intakes via Western style diets including sweetened beverages are below 500 mg per d but, when used as a sugar replacement, intake may reach 10 to 30 g per d depending on the food consumed. Due to its structural similarity with fructose, allulose uses the same transport and distribution pathways. But in contrast to fructose, the human genome does not encode for enzymes that are able to metabolise allulose leading to an almost complete renal excretion of the absorbed dose and near-to-zero energetic yield. However, in vitro studies have shown that certain bacteria such as Klebsiella pneumonia are able to utilise allulose as a substrate. This finding has been a subject of concern, since Klebsiella pneumoniae represents an opportunistic human pathogen. It therefore raised the question of whether a high dietary intake of allulose may cause an undesirable growth advantage for potentially harmful bacteria at mucosal sites such as the intestine or at systemic sites following invasive infection. In this brief review, we discuss the current state of science on these issues and define the research needs to better understand the fate of allulose and its metabolic and microbiological effects when ingested as a sugar substitute.

Associations between dietary patterns, FTO genotype and obesity in adults from seven European countries.

PURPOSE: High-fat and low-fibre discretionary food intake and FTO genotype are each associated independently with higher risk of obesity. However, few studies have investigated links between obesity and dietary patterns based on discretionary food intake, and the interaction effect of FTO genotype are unknown. Thus, this study aimed to derive dietary patterns based on intake of discretionary foods, saturated fatty acids (SFA) and fibre, and examine cross-sectional associations with BMI and waist circumference (WC), and interaction effects of FTO genotype. METHODS: Baseline data on 1280 adults from seven European countries were included (the Food4Me study). Dietary intake was estimated from a Food Frequency Questionnaire. Reduced rank regression was used to derive three dietary patterns using response variables of discretionary foods, SFA and fibre density. DNA was extracted from buccal swabs. Anthropometrics were self-measured. Linear regression analyses were used to examine associations between dietary patterns and BMI and WC, with an interaction for FTO genotype. RESULTS: Dietary pattern 1 (positively correlated with discretionary foods and SFA, and inversely correlated with fibre) was associated with higher BMI (ß:0.64; 95% CI 0.44, 0.84) and WC (ß:1.58; 95% CI 1.08, 2.07). There was limited evidence dietary pattern 2 (positively correlated with discretionary foods and SFA) and dietary pattern 3 (positively correlated with SFA and fibre) were associated with anthropometrics. FTO risk genotype was associated with higher BMI and WC, with no evidence of a dietary interaction. CONCLUSIONS: Consuming a dietary pattern low in discretionary foods and high-SFA and low-fibre foods is likely to be important for maintaining a healthy weight, regardless of FTO predisposition to obesity. TRIAL REGISTRATION: Clinicaltrials.gov NCT01530139. Registered 9 February 2012 https://clinicaltrials.gov/ct2/show/NCT01530139.

Personalised nutrition advice reduces intake of discretionary foods and beverages: findings from the Food4Me randomised controlled trial.

BACKGROUND: The effect of personalised nutrition advice on discretionary foods intake is unknown. To date, two national classifications for discretionary foods have been derived. This study examined changes in intake of discretionary foods and beverages following a personalised nutrition intervention using these two classifications. METHODS: Participants were recruited into a 6-month RCT across seven European countries (Food4Me) and were randomised to receive generalised dietary advice (control) or one of three levels of personalised nutrition advice (based on diet [L1], phenotype [L2] and genotype [L3]). Dietary intake was derived from an FFQ. An analysis of covariance was used to determine intervention effects at month 6 between personalised nutrition (overall and by levels) and control on i) percentage energy from discretionary items and ii) percentage contribution of total fat, SFA, total sugars and salt to discretionary intake, defined by Food Standards Scotland (FSS) and Australian Dietary Guidelines (ADG) classifications. RESULTS: Of the 1607 adults at baseline, n = 1270 (57% female) completed the intervention. Percentage sugars from FSS discretionary items was lower in personalised nutrition vs control (19.0 ± 0.37 vs 21.1 ± 0.65; P = 0.005). Percentage energy (31.2 ± 0.59 vs 32.7 ± 0.59; P = 0.031), percentage total fat (31.5 ± 0.37 vs 33.3 ± 0.65; P = 0.021), SFA (36.0 ± 0.43 vs 37.8 ± 0.75; P = 0.034) and sugars (31.7 ± 0.44 vs 34.7 ± 0.78; P < 0.001) from ADG discretionary items were lower in personalised nutrition vs control. There were greater reductions in ADG percentage energy and percentage total fat, SFA and salt for those randomised to L3 vs L2. CONCLUSIONS: Compared with generalised dietary advice, personalised nutrition advice achieved greater reductions in discretionary foods intake when the classification included all foods high in fat, added sugars and salt. Future personalised nutrition approaches may be used to target intake of discretionary foods. TRIAL REGISTRATION: Clinicaltrials.gov NCT01530139 . Registered 9 February 2012.

Diet and the gut microbiome: from hype to hypothesis.

Microbiome research in the last two decades has delivered as a key finding that the human intestine hosts a unique and complex ecosystem with many variables affecting the composition of the microbiota and in turn its function in metabolism and immune defence. Hundreds of external (environmental) factors have meanwhile been identified as significantly associated with bacterial biomass and diversity and, amongst these, diet is considered as a key determinant of microbial populations. However, dietary intervention studies, including those with fermentable substrates that have bulk effects on bowel functions, have revealed only very minor effects on overall microbiome composition and usually show only a very few species changing in population size. What that means in the context of hundreds of different species coexisting in competition or mutualism in the human colon is far from understood. This review addresses some of the current limits in research on diet effects by taking anatomical and physiological features of the intestine into consideration. It also provides some recommendations on future human studies needed to assess how the diet influences the microbiome and associated effects on metabolic health.

Characteristics of participants who benefit most from personalised nutrition: findings from the pan-European Food4Me randomised controlled trial.

Little is known about who would benefit from Internet-based personalised nutrition (PN) interventions. This study aimed to evaluate the characteristics of participants who achieved greatest improvements (i.e. benefit) in diet, adiposity and biomarkers following an Internet-based PN intervention. Adults (n 1607) from seven European countries were recruited into a 6-month, randomised controlled trial (Food4Me) and randomised to receive conventional dietary advice (control) or PN advice. Information on dietary intake, adiposity, physical activity (PA), blood biomarkers and participant characteristics was collected at baseline and month 6. Benefit from the intervention was defined as ≥5 % change in the primary outcome (Healthy Eating Index) and secondary outcomes (waist circumference and BMI, PA, sedentary time and plasma concentrations of cholesterol, carotenoids and omega-3 index) at month 6. For our primary outcome, benefit from the intervention was greater in older participants, women and participants with lower HEI scores at baseline. Benefit was greater for individuals reporting greater self-efficacy for 'sticking to healthful foods' and who 'felt weird if [they] didn't eat healthily'. Participants benefited more if they reported wanting to improve their health and well-being. The characteristics of individuals benefiting did not differ by other demographic, health-related, anthropometric or genotypic characteristics. Findings were similar for secondary outcomes. These findings have implications for the design of more effective future PN intervention studies and for tailored nutritional advice in public health and clinical settings.

Modifying effect of metabotype on diet-diabetes associations.

PURPOSE: Inter-individual metabolic differences may be a reason for previously inconsistent results in diet-diabetes associations. We aimed to investigate associations between dietary intake and diabetes for metabolically homogeneous subgroups ('metabotypes') in a large cross-sectional study. METHODS: We used data of 1517 adults aged 38-87 years from the German population-based KORA FF4 study (2013/2014). Dietary intake was estimated based on the combination of a food frequency questionnaire and multiple 24-h food lists. Glucose tolerance status was classified based on an oral glucose tolerance test in participants without a previous diabetes diagnosis using American Diabetes Association criteria. Logistic regression was applied to examine the associations between dietary intake and diabetes for two distinct metabotypes, which were identified based on 16 biochemical and anthropometric parameters. RESULTS: A low intake of fruits and a high intake of total meat, processed meat and sugar-sweetened beverages (SSB) were significantly associated with diabetes in the total study population. Stratified by metabotype, associations with diabetes remained significant for intake of total meat (OR 1.67, 95% CI 1.04-2.67) and processed meat (OR 2.23, 95% CI 1.24-4.04) in the metabotypes with rather favorable metabolic characteristics, and for intake of fruits (OR 0.83, 95% CI 0.68-0.99) and SSB (OR:1.21, 95% CI 1.09-1.35) in the more unfavorable metabotype. However, only the association between SSB intake and diabetes differed significantly by metabotype (p value for interaction = 0.01). CONCLUSIONS: Our findings suggest an influence of metabolic characteristics on diet-diabetes associations, which may help to explain inconsistent previous results. The causality of the observed associations needs to be confirmed in prospective and intervention studies.

Identification of essential amino acids for glucose transporter 5 (GLUT5)-mediated fructose transport.

Intestinal fructose uptake is mainly mediated by glucose transporter 5 (GLUT5/SLC2A5). Its closest relative, GLUT7, is also expressed in the intestine but does not transport fructose. For rat Glut5, a change of glutamine to glutamic acid at codon 166 (p.Q166E) has been reported to alter the substrate-binding specificity by shifting Glut5-mediated transport from fructose to glucose. Using chimeric proteins of GLUT5 and GLUT7, here we identified amino acid residues of GLUT5 that define its substrate specificity. The proteins were expressed in NIH-3T3 fibroblasts, and their activities were determined by fructose radiotracer flux. We divided the human GLUT5 sequence into 26 fragments and then replaced each fragment with the corresponding region in GLUT7. All fragments that yielded reduced fructose uptake were analyzed further by assessing the role of individual amino acid residues. Various positions in the first extracellular loop, in the fifth, seventh, eighth, ninth, and tenth transmembrane domains (TMDs), and in the regions between the ninth and tenth TMDs and tenth and 11th TMDs were identified as being important for proper fructose uptake. Although the p.Q167E change did not render the human protein into a glucose transporter, molecular dynamics simulations revealed a drastic change in the dynamics and a movement of the intracellular loop connecting the sixth and seventh TMDs, which covers the exit of the ligand. Finally, we generated a GLUT7-GLUT5 chimera consisting of the N-terminal part of GLUT7 and the C-terminal part of GLUT5. Although this chimera was inactive, we demonstrate fructose transport after introduction of four amino acids derived from GLUT5.

Myotube Protein Content Associates with Intracellular L-Glutamine Levels.

BACKGROUND/AIMS: Skeletal mass loss is reported in several catabolic conditions and it has been associated with a reduced intracellular L-glutamine content. We investigated the association of intracellular L-glutamine concentration with the protein content in skeletal muscle cells. METHODS: We cultivated C2C12 myotubes in the absence or presence of 2 (reference condition), 8 or 16 mM L-glutamine for 48 hours, and the variations in the contents of amino acids and proteins measured. We used an inhibitor of L-glutamine synthesis (L-methionine sulfoximine - MSO) to promote a further reduction in intracellular L-glutamine levels. Amino acids contents in cells and media were measured using LC-MS/MS. We measured changes in phosphorylated Akt, RP-S6, and 4E-BP1contents in the absence or presence of insulin by western blotting. RESULTS: Reduced intracellular L-glutamine concentration was associated with decreased protein content and increased protein breakdown. Low intracellular glutamine levels were also associated with decreased p-Akt contents in the presence of insulin. A further decrease in intracellular L-glutamine caused by glutamine synthetase inhibitor reduced protein content and levels of amino acids generated from glutamine metabolism and increased bAib still further. Cells exposed to high medium glutamine levels did not have any change in protein content but exhibited increased contents of the amino acids derived from L-glutamine metabolism. CONCLUSION: Intracellular L-glutamine levels per se play a role in the control of protein content in skeletal muscle myotubes.

Plasma metabolome analysis identifies distinct human metabotypes in the postprandial state with different susceptibility to weight loss-mediated metabolic improvements.

Health has been defined as the capability of the organism to adapt to challenges. In this study, we tested to what extent comprehensively phenotyped individuals reveal differences in metabolic responses to a standardized mixed meal tolerance test (MMTT) and how these responses change when individuals experience moderate weight loss. Metabolome analysis was used in 70 healthy individuals. with profiling of ∼300 plasma metabolites during an MMTT over 8 h. Multivariate analysis of plasma markers of fatty acid catabolism identified 2 distinct metabotype clusters (A and B). Individuals from metabotype B showed slower glucose clearance, had increased intra-abdominal adipose tissue mass and higher hepatic lipid levels when compared with individuals from metabotype A. An NMR-based urine analysis revealed that these individuals also to have a less healthy dietary pattern. After a weight loss of ∼5.6 kg over 12 wk, only the subjects from metabotype B showed positive changes in the glycemic response during the MMTT and in markers of metabolic diseases. Our study in healthy individuals demonstrates that more comprehensive phenotyping can reveal discrete metabotypes with different outcomes in a dietary intervention and that markers of lipid catabolism in plasma could allow early detection of the metabolic syndrome.-Fiamoncini, J., Rundle, M., Gibbons, H., Thomas, E. L., Geillinger-Kästle, K., Bunzel, D., Trezzi, J.-P., Kiselova-Kaneva, Y., Wopereis, S., Wahrheit, J., Kulling, S. E., Hiller, K., Sonntag, D., Ivanova, D., van Ommen, B., Frost, G., Brennan, L., Bell, J. Daniel, H. Plasma metabolome analysis identifies distinct human metabotypes in the postprandial state with different susceptibility to weight loss-mediated metabolic improvements.

Postprandial metabolic responses to ingestion of bovine glycomacropeptide compared to a whey protein isolate in prediabetic volunteers.

PURPOSE: Whey protein was shown to reduce blood glucose responses in humans and various other positive effects have been attributed to this protein. In contrast, studies using glycomacropeptide (GMP) as part of the whey fraction of bovine milk are rare. We, therefore, studied the postprandial responses to GMP administration in humans with impaired glucose tolerance compared to the effects of pure whey protein in a random design. METHODS: Fifteen prediabetic volunteers received on different occasions one of three test drinks containing 50 g of maltodextrin19 (MD19) alone or in combination with either 50 g GMP or 50 g whey protein isolate (WPI). Blood was collected over 4 h with analysis of blood glucose and hormones, gastric emptying rate as well as plasma amino- and fatty acids, ß-hydroxybutyrate and acylcarnitines. RESULTS: The WPI drink reduced the AUC of venous blood glucose compared to the MD19 drink in the prediabetic group by 11% (p = 0.0018) whereas GMP reduced the AUC by 18% (p < 0.0001), significantly different to the WPI drink (p = 0.0384). The reduction in blood glucose after the GMP drink was accompanied by a significantly lower AUC of insulin (- 34%) than for the WPI drink. Levels of C-peptide and of glucose insulinotropic polypeptide (GIP) were highly increased after the WPI drink over the MD19 control drink but remained in essence unaffected by the GMP. CONCLUSION: GMP reduced the glycemic response more potently than whey protein, whereas insulin output was less affected making GMP an interesting protein to control postprandial glucose responses.

Higher vegetable protein consumption, assessed by an isoenergetic macronutrient exchange model, is associated with a lower presence of overweight and obesity in the web-based Food4me European study.

The objective was to evaluate differences in macronutrient intake and to investigate the possible association between consumption of vegetable protein and the risk of overweight/obesity, within the Food4Me randomised, online intervention. Differences in macronutrient consumption among the participating countries grouped by EU Regions (Western Europe, British Isles, Eastern Europe and Southern Europe) were assessed. Relation of protein intake, within isoenergetic exchange patterns, from vegetable or animal sources with risk of overweight/obesity was assessed through the multivariate nutrient density model and a multivariate-adjusted logistic regression. A total of 2413 subjects who completed the Food4Me screening were included, with self-reported data on age, weight, height, physical activity and dietary intake. As success rates on reducing overweight/obesity are very low, form a public health perspective, the elaboration of policies for increasing intakes of vegetable protein and reducing animal protein and sugars, may be a method of combating overweight/obesity at a population level.

Bovine embryo elongation is altered due to maternal fatty acid supplementation.

The pre-implantation period is prone to embryonic losses in bovine. Embryo-maternal communication is crucial to support embryo development. Thereby, factors of the uterine fluid (UF) are of specific importance. The maternal diet can affect the UF composition. Since omega 3 fatty acids (omega 3 FA) are considered to be beneficial for reproduction, we investigated if dietary omega 3 FA affected factors in the UF related to embryo elongation. Angus heifers (n = 37) were supplemented with either 450 g of rumen-protected fish oil (omega 3 FA) or sunflower oil (omega 6 FA) for a period of 8 weeks. Following cycle synchronization and artificial insemination, the uteri were flushed post mortem to recover the embryos on day 15 of pregnancy. The UF and tissue samples of endometrium and corpus luteum (CL) were collected. Strikingly, the embryo elongation in the omega 3 group was enhanced compared to the omega 6 group. No differences were observed in uterine prostaglandins, even though the endometrial concentration of their precursor arachidonic acid was reduced in omega 3 compared to omega 6 heifers. The dietary FA neither led to differential expression of target genes in endometrium nor CL nor to a differential abundance of low-density lipoprotein cholesterol, cortisol or amino acids in the UF. Interestingly, the omega 3 group displayed a higher plasma progesterone concentration during luteal growth than the omega 6 group, possibly promoting embryo elongation. Further research should include an ovarian perspective to understand the functional link between dietary omega 3 FA and reproductive outcome.

Associations of vitamin D status with dietary intakes and physical activity levels among adults from seven European countries: the Food4Me study.

PURPOSE: To report the vitamin D status in adults from seven European countries and to identify behavioural correlates. METHODS: In total, 1075 eligible adult men and women from Ireland, Netherlands, Spain, Greece, UK, Poland and Germany, were included in the study. RESULTS: Vitamin D deficiency and insufficiency, defined as 25-hydroxy vitamin D3 (25-OHD3) concentration of <30 and 30-49.9 nmol/L, respectively, were observed in 3.3 and 30.6% of the participants. The highest prevalence of vitamin D deficiency was found in the UK and the lowest in the Netherlands (8.2 vs. 1.1%, P < 0.05). In addition, the prevalence of vitamin D insufficiency was higher in females compared with males (36.6 vs. 22.6%, P < 0.001), in winter compared with summer months (39.3 vs. 25.0%, P < 0.05) and in younger compared with older participants (36.0 vs. 24.4%, P < 0.05). Positive dose-response associations were also observed between 25-OHD3 concentrations and dietary vitamin D intake from foods and supplements, as well as with physical activity (PA) levels. Vitamin D intakes of ≥5 µg/day from foods and ≥5 µg/day from supplements, as well as engagement in ≥30 min/day of moderate- and vigorous-intensity PA were associated with higher odds (P < 0.05) for maintaining sufficient (≥50 nmol/L) 25-OHD3 concentrations. CONCLUSIONS: The prevalence of vitamin D deficiency varied considerably among European adults. Dietary intakes of ≥10 µg/day of vitamin D from foods and/or supplements and at least 30 min/day of moderate- and vigorous-intensity PA were the minimum thresholds associated with vitamin D sufficiency.

Amino Acid Transport Associated to Cluster of Differentiation 98 Heavy Chain (CD98hc) Is at the Cross-road of Oxidative Stress and Amino Acid Availability.

CD98hc functions as an amino acid (AA) transporter (together with another subunit) and integrin signaling enhancer. It is overexpressed in highly proliferative cells in both physiological and pathological conditions. CD98hc deletion induces strong impairment of cell proliferation in vivo and in vitro Here, we investigate CD98hc-associated AA transport in cell survival and proliferation. By using chimeric versions of CD98hc, the two functions of the protein can be uncoupled. Although recovering the CD98hc AA transport capacity restores the in vivo and in vitro proliferation of CD98hc-null cells, reconstitution of the integrin signaling function of CD98hc is unable to restore in vitro proliferation of those cells. CD98hc-associated transporters (i.e. xCT, LAT1, and y(+)LAT2 in wild-type cells) are crucial to control reactive oxygen species and intracellular AA levels, thus sustaining cell survival and proliferation. Moreover, in CD98hc-null cells the deficiency of CD98hc/xCT cannot be compensated, leading to cell death by ferroptosis. Supplementation of culture media with ß-mercaptoethanol rescues CD98hc-deficient cell survival. Under such conditions null cells show oxidative stress and intracellular AA imbalance and, consequently, limited proliferation. CD98hc-null cells also present reduced intracellular levels of branched-chain and aromatic amino acids (BCAAs and ARO AAs, respectively) and induced expression of peptide transporter 1 (PEPT1). Interestingly, external supply of dipeptides containing BCAAs and ARO AAs rescues cell proliferation and compensates for impaired uptake of CD98hc/LAT1 and CD98hc/y(+)LAT2. Our data establish CD98hc as a master protective gene at the cross-road of redox control and AA availability, making it a relevant therapeutic target in cancer.

Glycans in the intestinal peptide transporter PEPT1 contribute to function and protect from proteolysis.

Despite the fact that many membrane proteins carry extracellular glycans, little is known about whether the glycan chains also affect protein function. We recently demonstrated that the proton-coupled oligopeptide transporter 1 (PEPT1) in the intestine is glycosylated at six asparagine residues (N50, N406, N439, N510, N515, and N532). Mutagenesis-induced disruption of the individual N-glycosylation site N50, which is highly conserved among mammals, was detected to significantly enhance the PEPT1-mediated inward transport of peptides. Here, we show that for the murine protein the inhibition of glycosylation at sequon N50 by substituting N50 with glutamine, lysine, or cysteine or by replacing S52 with alanine equally altered PEPT1 transport kinetics in oocytes. Furthermore, we provide evidence that the uptake of [14C]glycyl-sarcosine in immortalized murine small intestinal (MODE-K) or colonic epithelial (PTK-6) cells stably expressing the PEPT1 transporter N50Q is also significantly increased relative to the wild-type protein. By using electrophysiological recordings and tracer flux studies, we further demonstrate that the rise in transport velocity observed for PEPT1 N50Q is bidirectional. In line with these findings, we show that attachment of biotin derivatives, comparable in weight with two to four monosaccharides, to the PEPT1 N50C transporter slows down the transport velocity. In addition, our experiments provide strong evidence that glycosylation of PEPT1 confers resistance against proteolytic cleavage by proteinase K, whereas a remarkable intrinsic stability against trypsin, even in the absence of N-linked glycans, was detected.NEW & NOTEWORTHY This study highlights the role of N50-linked glycans in modulating the bidirectional transport activity of the murine peptide transporter PEPT1. Electrophysiological and tracer flux measurements in Xenopus oocytes have shown that removal of the N50 glycans increases the maximal peptide transport rate in the inward and outward directions. This effect could be largely reversed by replacement of N50 glycans with structurally dissimilar biotin derivatives. In addition, N-glycans were detected to stabilize PEPT1 against proteolytic cleavage.

Determinants of postprandial plasma bile acid kinetics in human volunteers.

Bile acids (BA) are signaling molecules with a wide range of biological effects, also identified among the most responsive plasma metabolites in the postprandial state. We here describe this response to different dietary challenges and report on key determinants linked to its interindividual variability. Healthy men and women (n = 72, 62 ± 8 yr, mean ± SE) were enrolled into a 12-wk weight loss intervention. All subjects underwent an oral glucose tolerance test and a mixed-meal tolerance test before and after the intervention. BA were quantified in plasma by liquid chromatography-tandem mass spectrometry combined with whole genome exome sequencing and fecal microbiota profiling. Considering the average response of all 72 subjects, no effect of the successful weight loss intervention was found on plasma BA profiles. Fasting and postprandial BA profiles revealed high interindividual variability, and three main patterns in postprandial BA response were identified using multivariate analysis. Although the women enrolled were postmenopausal, effects of sex difference in BA response were evident. Exome data revealed the contribution of preselected genes to the observed interindividual variability. In particular, a variant in the SLCO1A2 gene, encoding the small intestinal BA transporter organic anion-transporting polypeptide-1A2 (OATP1A2), was associated with delayed postprandial BA increases. Fecal microbiota analysis did not reveal evidence for a significant influence of bacterial diversity and/or composition on plasma BA profiles. The analysis of plasma BA profiles in response to two different dietary challenges revealed a high interindividual variability, which was mainly determined by genetics and sex difference of host with minimal effects of the microbiota.NEW & NOTEWORTHY Considering the average response of all 72 subjects, no effect of the successful weight loss intervention was found on plasma bile acid (BA) profiles. Despite high interindividual variability, three main patterns in postprandial BA response were identified using multivariate analysis. A variant in the SLCO1A2 gene, encoding the small intestinal BA transporter organic anion-transporting polypeptide-1A2 (OATP1A2), was associated with delayed postprandial BA increases in response to both the oral glucose tolerance test and the mixed-meal tolerance test.

Reassessment of GLUT7 and GLUT9 as Putative Fructose and Glucose Transporters.

Although increased dietary fructose consumption is associated with metabolic impairments, the mechanisms and regulation of intestinal fructose absorption are poorly understood. GLUT5 is considered to be the main intestinal fructose transporter. Other GLUT family members, such as GLUT7 and GLUT9 are also expressed in the intestine and were shown to transport fructose and glucose. A conserved isoleucine-containing motif (NXI) was proposed to be essential for fructose transport capacity of GLUT7 and GLUT9 but also of GLUT2 and GLUT5. In assessing whether human GLUT2, GLUT5, GLUT7, and GLUT9 are indeed fructose transporters, we expressed these proteins in Xenopus laevis oocytes. Stably transfected NIH-3T3 fibroblasts were used as second expression system. In proving the role of the NXI motif, variants p.I322V of GLUT2 and p.I296V of GLUT5 were tested as well. Sugar transport was measured by radiotracer flux assays or by metabolomics analysis of cell extracts by GC-MS. Fructose and glucose uptakes by GLUT7 were not increased in both expression systems. In search for the physiological substrate of GLUT7, cells overexpressing the protein were exposed to various metabolite mixtures, but we failed to identify a substrate. Although urate transport by GLUT9 could be shown, neither fructose nor glucose transport was detectable. Fructose uptake was decreased by the GLUT2 p.I322V variant, but remained unaffected in the p.I296V GLUT5 variant. Thus, our work does not find evidence that GLUT7 or GLUT9 transport fructose or glucose or that the isoleucine residue determines fructose specificity. Rather, the physiological substrate of GLUT7 awaits to be discovered.