Relative abundance of the Prevotella genus within the human gut microbiota of elderly volunteers determines the inter-individual responses to dietary supplementation with wheat bran arabinoxylan-oligosaccharides.

BACKGROUND: The human colon is colonised by a dense microbial community whose species composition and metabolism are linked to health and disease. The main energy sources for colonic bacteria are dietary polysaccharides and oligosaccharides. These play a major role in modulating gut microbial composition and metabolism, which in turn can impact on health outcomes. RESULTS: We investigated the influence of wheat bran arabinoxylan oligosaccharides (AXOS) and maltodextrin supplements in modulating the composition of the colonic microbiota and metabolites in healthy adults over the age of 60. Male and female volunteers, (n = 21, mean BMI 25.2 ± 0.7 kg/m2) participated in the double-blind, cross over supplement study. Faecal samples were collected for analysis of microbiota, short chain fatty acids levels and calprotectin. Blood samples were collected to measure glucose, cholesterol and triglycerides levels. There was no change in these markers nor in calprotectin levels in response to the supplements. Both supplements were well-tolerated by the volunteers. Microbiota analysis across the whole volunteer cohort revealed a significant increase in the proportional abundance of faecal Bifidobacterium species (P ≤ 0.01) in response to AXOS, but not maltodextrin, supplementation. There was considerable inter-individual variation in the other bacterial taxa that responded, with a clear stratification of volunteers as either Prevotella-plus (n = 8; > 0.1% proportional abundance) or Prevotella-minus (n = 13; ≤0.1% proportional abundance) subjects founded on baseline sample profiles. There was a significant increase in the proportional abundance of both faecal Bifidobacterium (P ≤ 0.01) and Prevotella species (P ≤ 0.01) in Prevotella-plus volunteers during AXOS supplementation, while Prevotella and Bacteroides relative abundances showed an inverse relationship. Proportional abundance of 26 OTUs, including bifidobacteria and Anaerostipes hadrus, differed significantly between baseline samples of Prevotella-plus compared to Prevotella-minus individuals. CONCLUSIONS: The wheat bran AXOS supplementation was bifidogenic and resulted in changes in human gut microbiota composition that depended on the initial microbiota profile, specifically the presence or absence of Prevotella spp. as a major component of the microbiota. Our data therefore suggest that initial profiling of individuals through gut microbiota analysis should be considered important when contemplating nutritional interventions that rely on prebiotics. TRIAL REGISTRATION: Clinical trial registration number: NCT02693782 . Registered 29 February 2016 - Retrospectively registered, https://clinicaltrials.gov/ct2/show/NCT02693782?term=NCT02693782&rank=1.

Nutritional interest of dietary fiber and prebiotics in obesity: Lessons from the MyNewGut consortium.

The aim of EU project MyNewGut is to contribute to future public health-related recommendations supported by new insight in gut microbiome and nutrition-host relationship. In this Opinion Paper, we first revisit the concept of dietary fiber, taking into account their interaction with the gut microbiota. This paper also summarizes the main effects of dietary fibers with prebiotic properties in intervention studies in humans, with a particular emphasis on the effects of arabinoxylans and arabinoxylo-oligosaccharides on metabolic alterations associated with obesity. Based on the existing state of the art and future development, we elaborate the steps required to propose dietary guidelines related to dietary fibers, taking into account their interaction with the gut microbiota.

Impact of carbohydrate substrate complexity on the diversity of the human colonic microbiota.

The diversity of the colonic microbial community has been linked with health in adults and diet composition is one possible determinant of diversity. We used carefully controlled conditions in vitro to determine how the complexity and multiplicity of growth substrates influence species diversity of the human colonic microbiota. In each experiment, five parallel anaerobic fermenters that received identical faecal inocula were supplied continuously with single carbohydrates (either arabinoxylan-oligosaccharides (AXOS), pectin or inulin) or with a '3-mix' of all three carbohydrates, or with a '6-mix' that additionally contained resistant starch, ß-glucan and galactomannan as energy sources. Inulin supported less microbial diversity over the first 6 d than the other two single substrates or the 3- and 6-mixes, showing that substrate complexity is key to influencing microbiota diversity. The communities enriched in these fermenters did not differ greatly at the phylum and family level, but were markedly different at the species level. Certain species were promoted by single substrates, whilst others (such as Bacteroides ovatus, LEfSe P = 0.001) showed significantly greater success with the mixed substrate. The complex polysaccharides such as pectin and arabinoxylan-oligosaccharides promoted greater diversity than simple homopolymers, such as inulin. These findings suggest that dietary strategies intended to achieve health benefits by increasing gut microbiota diversity should employ complex non-digestible substrates and substrate mixtures.

Non-invasive continuous real-time in vivo analysis of microbial hydrogen production shows adaptation to fermentable carbohydrates in mice.

Real time in vivo methods are needed to better understand the interplay between diet and the gastrointestinal microbiota. Therefore, a rodent indirect calorimetry system was equipped with hydrogen (H2) and methane (CH4) sensors. H2 production was readily detected in C57BL/6J mice and followed a circadian rhythm. H2 production was increased within 12 hours after first exposure to a lowly-digestible starch diet (LDD) compared to a highly-digestible starch diet (HDD). Marked differences were observed in the faecal microbiota of animals fed the LDD and HDD diets. H2 was identified as a key variable explaining the variation in microbial communities, with specific taxa (including Bacteroides and Parasutterella) correlating with H2 production upon LDD-feeding. CH4 production was undetectable which was in line with absence of CH4 producers in the gut. We conclude that real-time in vivo monitoring of gases provides a non-invasive time-resolved system to explore the interplay between nutrition and gut microbes in a mouse model, and demonstrates potential for translation to other animal models and human studies.

Substitution of Corn Starch with Resistant Starch Type 4 in a Breakfast Bar Decreases Postprandial Glucose and Insulin Responses: A Randomized, Controlled, Crossover Study.

BACKGROUND: Resistant starches type 4 (RS4) are chemically modified starches that are resistant to digestion by human enzymes. OBJECTIVE: We aimed to test our hypothesis that replacement of standard starch with RS4 in a baked breakfast bar would decrease postprandial glycemic and insulinemic responses in healthy adults. METHODS: In this double-blind, randomized crossover study, 21 healthy adults [10 men; 20-45 y old; BMI (kg/m2): 19.3-27.0] consumed a baked breakfast bar containing tapioca-based RS4 (Actistar 75330; Cargill, Inc.) or a macronutrient-matched control bar, delivering 32 g and 4 g of dietary fiber, respectively. Primary outcome was the incremental area under the curve (iAUC0-120 min) for postprandial capillary glucose. Other outcomes included postprandial serum insulin iAUC0-120 min, glucose and insulin maximum concentration (Cmax), and time to Cmax (Tmax). RESULTS: Median glucose iAUC0-120 min was 22% lower (P < 0.05) and median insulin iAUC0-120 min was 37% lower (P < 0.05) after consumption of the RS4 food compared with the control food. Glucose and insulin Cmax and Tmax were not significantly different (P > 0.05) between foods. CONCLUSION: The results suggest that replacement of standard starch with tapioca-based RS4 is a practical approach for reducing available carbohydrate in products and achieving postprandial blood glucose management. This trial was registered at clinicaltrials.gov as NCT03239288.

Evaluation of the gastrointestinal tolerability of corn starch fiber, a novel dietary fiber, in two independent randomized, double-blind, crossover studies in healthy men and women.

Two independent clinical studies were conducted to compare the gastrointestinal (GI) tolerability of corn starch fiber, a novel dietary fiber, at up to 50 g/day (single-dose study) or 90 g/day (multiple-serving study) with a negative control (no fiber) and a positive control (50 or 90 g polydextrose, for single- and multiple-serving studies, respectively) in generally healthy study volunteers. Flatulence and borborygmus were the primary symptoms reported at the higher doses of corn starch fiber and for the positive control interventions. Bowel movements were increased over 48 h with corn starch fiber at 90 g. Thresholds for mild GI effects were established at 30 g as a single dose and 60 g as multiple servings spread over the day. Other than moderate abdominal pain and mild increased appetite in one subject at 90-g corn starch fiber, no test article-related adverse events were reported.

Transcriptional analysis of the Arabidopsis ovule by massively parallel signature sequencing.

The life cycle of flowering plants alternates between a predominant sporophytic (diploid) and an ephemeral gametophytic (haploid) generation that only occurs in reproductive organs. In Arabidopsis thaliana, the female gametophyte is deeply embedded within the ovule, complicating the study of the genetic and molecular interactions involved in the sporophytic to gametophytic transition. Massively parallel signature sequencing (MPSS) was used to conduct a quantitative large-scale transcriptional analysis of the fully differentiated Arabidopsis ovule prior to fertilization. The expression of 9775 genes was quantified in wild-type ovules, additionally detecting >2200 new transcripts mapping to antisense or intergenic regions. A quantitative comparison of global expression in wild-type and sporocyteless (spl) individuals resulted in 1301 genes showing 25-fold reduced or null activity in ovules lacking a female gametophyte, including those encoding 92 signalling proteins, 75 transcription factors, and 72 RNA-binding proteins not reported in previous studies based on microarray profiling. A combination of independent genetic and molecular strategies confirmed the differential expression of 28 of them, showing that they are either preferentially active in the female gametophyte, or dependent on the presence of a female gametophyte to be expressed in sporophytic cells of the ovule. Among 18 genes encoding pentatricopeptide-repeat proteins (PPRs) that show transcriptional activity in wild-type but not spl ovules, CIHUATEOTL (At4g38150) is specifically expressed in the female gametophyte and necessary for female gametogenesis. These results expand the nature of the transcriptional universe present in the ovule of Arabidopsis, and offer a large-scale quantitative reference of global expression for future genomic and developmental studies.

A single-chain tetradomain glycoprotein hormone analog elicits multiple hormone activities in vivo.

We previously demonstrated that genetically linking one or more of the glycoprotein hormone-specific beta subunit genes to the common alpha subunit resulted in single-chain analogues that were bioactive in vitro. The ability of such large structures to bind their cognate receptors with high affinity supported the hypothesis that extensive flexibility exists between the ligand and receptor to establish a functional complex. To further characterize the extent of this conformational flexibility, we engineered a single-chain analogue that consists of sequentially linked thyroid-stimulating hormone (TSH) beta, follicle-stimulating hormone (FSH) beta, and chorionic gonadotropin (CG) beta subunits to the alpha subunit and expressed this chimera in transfected CHO (Chinese hamster ovary) cells. Because the four subunits are genetically linked and expressed as a single-chain, this analogue presumably lacks significant native structural features of the individual heterodimers. However, it exhibited FSH, CG, and TSH activities in vitro. Here, we test whether this nonnative structure would be stable in vivo and thus biologically active. Using a variety of bioassay protocols, we demonstrate that the analogue elicits multihormone activities when injected in vivo. First, treatment with the analogue caused increases in ovarian and uterine weights and resulted in elevated serum estradiol. Second, the analogue-stimulated ovarian follicle growth and pharmacologically rescued in vivo FSH deficiency similar to recombinant human FSH or equine CG (eCG) as confirmed by induction of aromatase in the ovaries of FSHbeta knockout mice. Third, in a superovulation protocol, when primed with eCG, the analogue elicited a dose-dependent ovulatory response comparable with that by native heterodimeric human CG. Finally, the analogue-stimulated thyroxin production in hypothyroid mice similar to the pituitary-derived human TSH standard. Based on these data, we conclude that a single-chain tetradomain glycoprotein hormone analogue, despite its presumed altered conformation, is stable and biologically active in vivo. Our results establish the permissiveness and conformational plasticity with which the glycoprotein hormones are recognized in vivo by their target cell receptors.

A single-chain bifunctional gonadotropin analog is secreted from Chinese hamster ovary cells as two distinct bioactive species.

One of the major developments in exploring structure activity relationships of the glycoprotein hormone family was the genetic engineering of single chains comprised of the common alpha subunit and one or more of the hormone-specific beta subunits tandemly arranged. These studies indicate that there is a structural permissiveness in the quaternary relationships between the subunits and biological activity. However, the conformational relationships between the ligand and the receptor are unclear. Bifunctional triple-domain analogs represent an ideal model to address this issue. Does a single molecule possess the ability to simultaneously interact with both specific receptors or are there two functionally distinct species in the chimeric population? Here we show, using a preadsorption protocol comprised of Chinese hamster ovary cells expressing either the luteinizing hormone (LH)/chorionic gonadotropin (CG) or follicle-stimulating hormone (FSH) receptor, that at least two distinct bioactive populations of the dually active triple-domain chimera FSHbeta-CGbeta-alpha are synthesized, each corresponding to a single activity (CG or FSH). Furthermore, we show that these bioactive populations form distinct stable heterodimer-like contacts. That there is not a single biologically active species formed during synthesis of the chimera implies that in vivo the heterodimer exists in multiple conformations and is not a static rigid molecule.

Unmasking a new recognition signal for O-linked glycosylation in the chorionic gonadotropin beta subunit.

hCGbeta subunit is distinguished among the other members of the family of the glycoprotein hormones by the presence of four serine O-linked oligosaccharide units in the last 25 amino acids. This carboxy terminal peptide (CTP) influences the intracellular behavior of the subunit and is important for maintaining the biological half-life of hCG. To examine how the O-linked oligosaccharides affect the metabolic behavior of hCG, we generated a CGbeta mutant devoid of the native O-linked acceptor sites. An alternative site not used in the native subunit was glycosylated and the structure of this oligosaccharide differed from the wild-type O-linked carbohydrates. This glycosylation occurred at serine 130 in the CTP and in contrast to the wild type O-linked oligosaccharides, sialic acid is a major component of the alternatively linked carbohydrate. The data show that deleting the native acceptor sites exposes a new site for O-glycosylation and promotes a differential intracellular processing of the beta subunit. These results support the hypothesis that the CTP participates in the folding of the newly synthesized subunit, which is manifested by the post-translational changes reported here.

Thyrotropin, follitropin, and chorionic gonadotropin expressed as a single multifunctional unit reveal remarkable permissiveness in receptor-ligand interactions.

The glycoprotein hormones [chorionic gonadotropin (CG), FSH, LH, and TSH] are composed of a common alpha-subunit and a hormone-specific beta-subunit. Subunit assembly is vital to the in vivo function of these hormones. However, recent in vitro studies using double domain (beta-alpha) and triple domain (beta-beta-alpha) single chains have shown that gonadotropin receptor recognition can accommodate conformationally modified ligands. To investigate the extent of flexibility of ligand-receptor interactions, we constructed a single chain tetramer containing three different beta-subunits (TSHbeta, FSHbeta, and CGbeta) and a single alpha-subunit. This analog was inefficiently secreted from transfected Chinese hamster ovary cells, but surprisingly, the protein exhibited all activities comparable to the corresponding heterodimers. Because the alpha-subunit presumably cannot form the entire array of heterodimeric contacts with all beta-subunits simultaneously in the tetra-domain analog, the data show that the complete quaternary subunit-subunit interactions are essential for the efficient intracellular trafficking of the glycoprotein hormones, but not for receptor recognition. From an evolutionary perspective, the organization of such a multifunctional analog is consistent with the hypothesis that glycoprotein hormone genes were originally linked in tandem and subsequently evolved as independent genes. Our results also indicate that both gonadal and thyroid stimulatory functions can be combined in a unique analog.

Evolution of lutropin to chorionic gonadotropin generates a specific routing signal for apical release in vivo.

One of the fundamental differences among mammals is the mechanism of maintaining the corpus luteum of pregnancy. Placentation in primates is associated with the production of the glycoprotein hormone chorionic gonadotropin (CG), which is secreted into the maternal serum and stimulates progesterone synthesis from the corpus luteum, which is essential for early development of the embryo. CG together with the pituitary hormones lutropin (LH), follitropin, and thyrotropin constitute the family of glycoprotein hormones comprised of a common alpha subunit and a hormone-specific beta subunit. The LHbeta and CGbeta subunits share 85% amino acid sequence identity, and functionally LH and CG are interchangeable. CGbeta evolved by a recent gene duplication event from the LHbeta locus, and despite the close relationship between them, their modes of secretion are quite different. CG release from the placenta is apically directed, whereas LH is released from the basal side of the cell, and the determinant(s) for this redirected trafficking are unknown. Here, using the polarized Madin-Darby canine kidney (MDCK) cell line, we provide evidence for the molecular basis of the different secretory patterns of LH and CG in vivo. The apical targeting of CG is programmed by a carboxyl-terminal sequence, which encodes a novel sorting signal. It is also apparent that the presence of the O-linked oligosaccharides in the CTP sequence contributes to this apical routing. The CTP, which is absent in LH, redirects CG to the maternal serum and permits the unique arrangement for primate placentation. Our data also show that the MDCK cells can distinguish the different secretory pathways for the gonadotropins and will be a valuable model for elucidating the determinants associated with the unique sorting of these functionally related hormones.