ObjectiveContrary to the long-standing prerequisite of inducing selective (ie, bifidogenic) effects, recent findings suggest that prebiotic interventions lead to ecosystem-wide microbiota shifts. Yet, a comprehensive characterisation of this process is still lacking. Here, we apply 16S rDNA microbiota profiling and matching (gas chromatography mass spectrometry) metabolomics to assess the consequences of inulin fermentation both on the composition of the colon bacterial ecosystem and faecal metabolites profiles.DesignFaecal samples collected during a double-blind, randomised, cross-over intervention study set up to assess the effect of inulin consumption on stool frequency in healthy adults with mild constipation were analysed. Faecal microbiota composition and metabolite profiles were linked to the study's clinical outcome as well as to quality-of-life measurements recorded.ResultsWhile faecal metabolite profiles were not significantly altered by inulin consumption, our analyses did detect a modest effect on global microbiota composition and specific inulin-induced changes in relative abundances of Anaerostipes, Bilophila and Bifidobacterium were identified. The observed decrease in Bilophila abundances following inulin consumption was associated with both softer stools and a favourable change in constipation-specific quality-of-life measures.ConclusionsEcosystem-wide analysis of the effect of a dietary intervention with prebiotic inulin-type fructans on the colon microbiota revealed that this effect is specifically associated with three genera, one of which (Bilophila) representing a promising novel target for mechanistic research.Trial registration numberNCT02548247.

Metabolic challenge protocols, such as the oral glucose tolerance test, can uncover early alterations in metabolism preceding chronic diseases. Nevertheless, most metabolomics data accessible today reflect the fasting state. To analyze the dynamics of the human metabolome in response to environmental stimuli, we submitted 15 young healthy male volunteers to a highly controlled 4 d challenge protocol, including 36 h fasting, oral glucose and lipid tests, liquid test meals, physical exercise, and cold stress. Blood, urine, exhaled air, and breath condensate samples were analyzed on up to 56 time points by MS- and NMR-based methods, yielding 275 metabolic traits with a focus on lipids and amino acids. Here, we show that physiological challenges increased interindividual variation even in phenotypically similar volunteers, revealing metabotypes not observable in baseline metabolite profiles; volunteer-specific metabolite concentrations were consistently reflected in various biofluids; and readouts from a systematic model of β-oxidation (e.g., acetylcarnitine/palmitylcarnitine ratio) showed significant and stronger associations with physiological parameters (e.g., fat mass) than absolute metabolite concentrations, indicating that systematic models may aid in understanding individual challenge responses. Due to the multitude of analytical methods, challenges and sample types, our freely available metabolomics data set provides a unique reference for future metabolomics studies and for verification of systems biology models.

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Intake of protein immediately after exercise stimulates protein synthesis but improved recovery of performance is not consistently observed. The primary aim of the present study was to compare performance 18 h after exhaustive cycling in a randomized diet-controlled study (175 kJ·kg-1 during 18 h) when subjects were supplemented with protein plus carbohydrate or carbohydrate only in a 2-h window starting immediately after exhaustive cycling. The second aim was to investigate the effect of no nutrition during the first 2 h and low total energy intake (113 kJ·kg-1 during 18 h) on performance when protein intake was similar. Eight endurance-trained subjects cycled at 237±6 Watt (~72% VO2max) until exhaustion (TTE) on three occasions, and supplemented with 1.2 g carbohydrate·kg-1·h-1 (CHO), 0.8 g carbohydrate + 0.4 g protein·kg-1·h-1 (CHO+PRO) or placebo without energy (PLA). Intake of CHO+PROT increased plasma glucose, insulin, and branch chained amino acids, whereas CHO only increased glucose and insulin. Eighteen hours later, subjects performed another TTE at 237±6 Watt. TTE was increased after intake of CHO+PROT compared to CHO (63.5±4.4 vs 49.8±5.4 min; p<0.05). PLA reduced TTE to 42.8±5.1 min (p<0.05 vs CHO). Nitrogen balance was positive in CHO+PROT, and negative in CHO and PLA. In conclusion, performance was higher 18 h after exhaustive cycling with intake of CHO+PROT compared to an isocaloric amount of carbohydrate during the first 2 h post exercise. Intake of a similar amount of protein but less carbohydrate during the 18 h recovery period reduced performance.

In humans, plasma amino acid concentrations of branched-chain amino acids (BCAA) and aromatic amino acids (AAA) increase in states of obesity, insulin resistance and diabetes. We here assessed whether these putative biomarkers can also be identified in two different obesity and diabetic mouse models. C57BL/6 mice with diet-induced obesity (DIO) mimic the metabolic impairments of obesity in humans characterized by hyperglycemia, hyperinsulinemia and hepatic triglyceride accumulation. Mice treated with streptozotocin (STZ) to induce insulin deficiency were used as a type 1 diabetes model. Plasma amino acid profiling of two high fat (HF) feeding trials revealed that citrulline and ornithine concentrations are elevated in obese mice, while systemic arginine bioavailability (ratio of plasma arginine to ornithine + citrulline) is reduced. In skeletal muscle, HF feeding induced a reduction of arginine levels while citrulline levels were elevated. However, arginine or citrulline remained unchanged in their key metabolic organs, intestine and kidney. Moreover, the intestinal conversion of labeled arginine to ornithine and citrulline in vitro remained unaffected by HF feeding excluding the intestine as prime site of these alterations. In liver, citrulline is mainly derived from ornithine in the urea cycle and DIO mice displayed reduced hepatic ornithine levels. Since both amino acids share an antiport mechanism for mitochondrial import and export, elevated plasma citrulline may indicate impaired hepatic amino acid handling in DIO mice. In the insulin deficient mice, plasma citrulline and ornithine levels also increased and additionally these animals displayed elevated BCAA and AAA levels like insulin resistant and diabetic patients. Therefore, type 1 diabetic mice but not DIO mice show the “diabetic fingerprint” of plasma amino acid changes observed in humans. Additionally, citrulline may serve as an early indicator of the obesity-dependent metabolic impairments.

Non-alcoholic fatty liver disease (NAFLD) results from increased hepatic lipid accumulation and steatosis, and is closely linked to liver one-carbon (C1) metabolism. We assessed in C57BL6/N mice whether NAFLD induced by a high-fat (HF) diet over 8 weeks can be reversed by additional 4 weeks of a dietary methyl-donor supplementation (MDS). MDS in the obese mice failed to reverse NAFLD, but prevented the progression of hepatic steatosis associated with major changes in key hepatic C1-metabolites, e.g. S-adenosyl-methionine and S-adenosyl-homocysteine. Increased phosphorylation of AMPK-α together with enhanced β-HAD activity suggested an increased flux through fatty acid oxidation pathways. This was supported by concomitantly decreased hepatic free fatty acid and acyl-carnitines levels. Although HF diet changed the hepatic phospholipid pattern, MDS did not. Our findings suggest that dietary methyl-donors activate AMPK, a key enzyme in fatty acid β-oxidation control, that mediates increased fatty acid utilization and thereby prevents further hepatic lipid accumulation.

Glutamine, the most abundant amino acid in mammals, is critical for cell and organ functions. Its metabolism depends on the ability of cells to take up or release glutamine by transporters located in the plasma membrane. Several solute carrier (SLC) families transport glutamine, but the SLC38 family has been thought to be mostly responsible for glutamine transport. We demonstrate that despite the large number of glutamine transporters, the loss of Snat3/Slc38a3 glutamine transporter has a major impact on the function of organs expressing it. Snat3 mutant mice were generated by N-ethyl-N-nitrosurea (ENU) mutagenesis and showed stunted growth, altered amino acid levels, hypoglycemia, and died around 20 days after birth. Hepatic concentrations of glutamine, glutamate, leucine, phenylalanine, and tryptophan were highly reduced paralleled by downregulation of the mTOR pathway possibly linking reduced amino acid availability to impaired growth and glucose homeostasis. Snat3-deficient mice had altered urea levels paralleled by dysregulation of the urea cycle, gluconeogenesis, and glutamine synthesis. Mice were ataxic with higher glutamine but reduced glutamate and gamma-aminobutyric acid (GABA) levels in brain consistent with a major role of Snat3 in the glutamine-glutamate cycle. Renal ammonium excretion was lower, and the expression of enzymes and amino acid transporters involved in ammoniagenesis were altered. Thus, SNAT3 is a glutamine transporter required for amino acid homeostasis and determines critical functions in various organs. Despite the large number of glutamine transporters, loss of Snat3 cannot be compensated, suggesting that this transporter is a major route of glutamine transport in the liver, brain, and kidney. ABSTRACTGlutamine, the most abundant amino acid in mammals, is critical for cell and organ functions. Its metabolism depends on the ability of cells to take up or release glutamine by transporters located in the plasma membrane. Several solute carrier (SLC) families transport glutamine, but the SLC38 family has been thought to be mostly responsible for glutamine transport. We demonstrate that despite the large number of glutamine transporters, loss of the Snat3/Slc38a3 glutamine transporter has a major impact on the function of organs expressing it. Snat3 mutant mice were generated by N-ethyl-N-nitrosurea (ENU) mutagenesis and showed stunted growth, altered amino acid levels, hypoglycemia, and died around 20 days after birth. Hepatic concentrations of glutamine, glutamate, leucine, phenylalanine, and tryptophan were highly reduced paralleled by downregulation of the mTOR pathway possibly linking reduced amino acid availability to impaired growth and glucose homeostasis. Snat3 deficient mice had altered urea levels paralleled by dysregulation of the urea cycle, gluconeogenesis, and glutamine synthesis. Mice were ataxic with higher glutamine but reduced glutamate and GABA levels in brain consistent with a major role of Snat3 in the glutamine-glutamate cycle. Renal ammonium excretion ...

Obesity frequently leads to insulin resistance and the development of hepatic steatosis. To characterize the molecular changes that promote hepatic steatosis, transcriptomics, proteomics, and metabolomics technologies were applied to liver samples from C57BL/6J mice obtained from two independent intervention trials. After 12 wk of high-fat feeding the animals became obese, hyperglycemic, and insulin resistant, had elevated levels of blood cholesterol and VLDL, and developed hepatic steatosis. Nutrigenomic analysis revealed alterations of key metabolites and enzyme transcript levels of hepatic one-carbon metabolism and related pathways. The hepatic oxidative capacity and the lipid milieu were significantly altered, which may play a key role in the development of insulin resistance. Additionally, high choline levels were observed after the high-fat diet. Previous studies have linked choline levels with insulin resistance and hepatic steatosis in conjunction with changes of certain metabolites and enzyme levels of one-carbon metabolism. The present results suggest that the coupling of high levels of choline and low levels of methionine plays an important role in the development of insulin resistance and liver steatosis. In conclusion, the complexities of the alterations induced by high-fat feeding are multifactorial, indicating that the interplay between several metabolic pathways is responsible for the pathological consequences.