Low–molecular-weight metabolites produced by intestinal microbiota play a direct role in health and disease. In this study, we analyzed the colonic luminal metabolome using capillary electrophoresis mass spectrometry with time-of-flight (CE-TOFMS) —a novel technique for analyzing and differentially displaying metabolic profiles— in order to clarify the metabolite profiles in the intestinal lumen. CE-TOFMS identified 179 metabolites from the colonic luminal metabolome and 48 metabolites were present in significantly higher concentrations and/or incidence in the germ-free (GF) mice than in the Ex-GF mice (p < 0.05), 77 metabolites were present in significantly lower concentrations and/or incidence in the GF mice than in the Ex-GF mice (p < 0.05), and 56 metabolites showed no differences in the concentration or incidence between GF and Ex-GF mice. These indicate that intestinal microbiota highly influenced the colonic luminal metabolome and a comprehensive understanding of intestinal luminal metabolome is critical for clarifying host-intestinal bacterial interactions.

Prevention of quality of life (QOL) deterioration is associated with the inhibition of geriatric diseases and the regulation of brain function. However, no substance is known that prevents the aging of both body and brain. It is known that polyamine concentrations in somatic tissues (including the brain) decrease with increasing age, and polyamine-rich foods enhance longevity in yeast, worms, flies, and mice, and protect flies from age-induced memory impairment. A main source of exogenous polyamines is the intestinal lumen, where they are produced by intestinal bacteria. We found that arginine intake increased the concentration of putrescine in the colon and increased levels of spermidine and spermine in the blood. Mice orally administered with arginine in combination with the probiotic bifidobacteria LKM512 long-term showed suppressed inflammation, improved longevity, and protection from age-induced memory impairment. This study shows that intake of arginine and LKM512 may prevent aging-dependent declines in QOL via the upregulation of polyamines.

Metabolomics is an emerging technology that reveals homeostatic imbalances in biological systems. Global determination of metabolite concentrations in body fluid and tissues provides novel anatomical aspects of pathological conditions that cannot be obtained from target-specific measurements. Here, we characterised metabolic imbalance in Watanabe heritable hyperlipidaemic rabbits as a model of hypercholesterolaemia. Using a mass spectrometry-based system, we measured a total of 335 metabolites in plasma and tissues (liver, aorta, cardiac muscle, and brain) from WHHL and healthy control rabbits. From the comparison between two metabolomic profiles, pathophysiological features including glutathione and phosphatidylcholine metabolism indicated the occurrence of oxidative stress in several tissues. Especially for the liver, imbalanced purine catabolism shed light on the transcriptional activation of xanthine oxidase, which is thought to act in absorbing or possibly triggering oxidative stress. We also applied this system to assess the therapeutic effects of simvastatin administration. After the treatment, a portion of the metabolomic features in pathological conditions showed alterations suggesting restoration of metabolism to the healthy condition. These changes were considered to be due to the pleiotropic action of statin, including antioxidant effects, rather than its main inhibitory action on cholesterol biosynthesis.

Recent studies suggest that intestinal microbiota influences gut-brain communication. In this study, we aimed to clarify the influence of intestinal microbiota on cerebral metabolism. We analyzed the cerebral metabolome of germ-free (GF) mice and Ex-GF mice, which were inoculated with suspension of feces obtained from specific pathogen-free mice, using capillary electrophoresis with time-of-flight mass spectrometry (CE-TOFMS). CE-TOFMS identified 196 metabolites from the cerebral metabolome in both GF and Ex-GF mice. The concentrations of 38 metabolites differed significantly (p < 0.05) between GF and Ex-GF mice. Approximately 10 of these metabolites are known to be involved in brain function, whilst the functions of the remainder are unclear. Furthermore, we observed a novel association between cerebral glycolytic metabolism and intestinal microbiota. Our work shows that cerebral metabolites are influenced by normal intestinal microbiota through the microbiota-gut-brain axis, and indicates that normal intestinal microbiota closely connected with brain health and disease, development, attenuation, learning, memory, and behavior.

ADP-ribose pyrophosphatase (ADPRase) catalyzes the divalent metal ion-dependent hydrolysis of ADP-ribose to ribose 5 -phosphate and AMP. This enzyme plays a key role in regulating the intracellular ADP-ribose levels, and prevents nonenzymatic ADP-ribosylation. To elucidate the pyrophosphatase hydrolysis mechanism employed by this enzyme, structural changes occurring on binding of substrate, metal and product were investigated using crystal structures of ADPRase from an extreme thermophile, Thermus thermophilus HB8. Seven structures were determined, including that of the free enzyme, the Zn 2؉ -bound enzyme, the binary complex with ADP-ribose, the ternary complexes with ADPribose and Zn 2؉ or Gd 3؉ , and the product complexes with AMP and Mg 2؉ or with ribose 5 -phosphate and Zn 2؉ . The structural and functional studies suggested that the ADP-ribose hydrolysis pathway consists of four reaction states: bound with metal (I), metal and substrate (II), metal and substrate in the transition state (III), and products (IV). In reaction state II, Glu-82 and Glu-70 abstract a proton from a water molecule. This water molecule is situated at an ideal position to carry out nucleophilic attack on the adenosyl phosphate, as it is 3.6 Å away from the target phosphorus and almost in line with the scissile bond.Nudix pyrophosphatases are widely distributed in nature and share a highly conserved amino acid sequence motif called the "Nudix motif " (GX 5 EX 7 REUXEEXGU, where U is one of the bulky hydrophobic amino acids I, L, or V), which adopts a unique loop-helix-loop structure (1). Enzymes in this family catalyze the hydrolysis of nucleoside diphosphates, linked to another moiety x. Their postulated role is to control the cellular concentration of toxic nucleoside diphosphate derivatives or physiological metabolites, accumulation of which could be harmful (1). ADP-ribose (ADPR) 1 is one such diphosphate derivative, which is produced enzymatically as part of the turnover of NAD ϩ , cyclic ADPR, ADP-ribosylated proteins, and poly-ADP-ribosylated proteins. Although certain proteins are posttranslationally modified by ADPR, high intracellular levels of ADPR could result in nonenzymatic ADP-ribosylation. This is a deleterious process that inactivates enzymes and could interfere with the recognition of enzymatic ADP-ribosylation (2). ADPR pyrophosphatases (ADPRases) catalyze the hydrolysis of ADPR to AMP and ribose 5Ј-phosphate to prevent ADPR accumulation.ADPRase activity has been detected in all three kingdoms (3-7), but the specificity for ADPR over other substrates and the selectivity of metal ions required for activity vary between species. The mechanism underlying the different substrate specificity and the metal dependence is unknown at both the structural and functional levels. Elucidation of these properties requires the study of ADPRases from numerous sources.In this article, we investigated the catalytic mechanism of ADPRase from an extreme thermophile, Thermus thermophilus HB8 (TtADPRase). In general, proteins isolated fr...

A major bacterial alarmone, guanosine 3,5-bispyrophosphate (ppGpp), controls cellular growth under conditions of nutritional starvation. For most bacteria, intracellular ppGpp levels are tightly controlled by the synthesis/degradation cycle of RelA and SpoT activities. This study shows a novel ppGpp regulatory protein governing the cellular growth of Thermus thermophilus, Ndx8, a member of the Nudix pyrophosphatase family that degrades ppGpp to yield guanosine 3,5-bisphosphate. The ndx8-null mutant strain exhibited early stage growth arrest accompanied by the stationary phase-specific morphologies and global transcriptional modulation under nutritionally defined conditions. Several possible substrate compounds of Ndx8, which specifically accumulated in the ndx8 mutant cells, were identified by employing a capillary electrophoresis time-of-flight mass spectrometry-based metabolomics approach. Among them, the hydrolytic activity of Ndx8 for ppGpp was significant not only in vitro but also in vivo. Finally, the elimination of ppGpp synthetic activity suppressed the observed phenotype of the ndx8 mutation, suggesting that the function of Ndx8 as a growth regulator is involved in ppGpp accumulation, which is thought to act as a trigger of the growth phase transition. These results suggest a novel mechanism of ppGpp-mediated growth control by the functional relay between Ndx8 and SpoT activity as ppGpp scavengers.The stringent response, which is a pleiotropic adaptation to nutritional starvation and stress, is broadly conserved in bacteria and plant chloroplasts (1, 2). This physiological control is dominated by a rapid synthesis/degradation cycle of the stringent alarmones guanosine 3Ј-pyrophosphate 5Ј-triphosphate (pppGpp) 4 or guanosine 3Ј,5Ј-bispyrophosphate (ppGpp) ( Fig. 1) (3). In Escherichia coli grown under amino acid depletion, pppGpp is synthesized from ATP and GTP by ribosome-associated RelA activity and subsequently converted to ppGpp by the activity of 5Ј-nucleotidase (4). Besides this pathway, ppGpp is also directly synthesized from GDP by the activity of RelA. Thereafter, ppGpp is degraded to GDP and pyrophosphate by the activity of SpoT, which is a RelA homolog with reciprocal activities for ppGpp degradation and synthesis (5, 6). For several bacteria, SpoT physiologically acts as both the ppGpp synthetase and hydrolase and is known as the Rel/Spo homolog. The metabolism of these alarmone nucleotides is understood based on the function of these two enzymes, whereas recent studies have identified alternative players acting as ppGpp synthetase in bacteria (7,8). On the other hand, for the degradation of ppGpp, previous studies reported that SpoT-independent (p)ppGpp degradation activity exists in bacteria, whereas the biological meaning(s) and enzymatic mechanism(s) of these alternative pathways have not been characterized in any organisms (9 -12).The Nudix hydrolases constitute a large family of proteins that share a highly conserved amino acid sequence, the so-called "Nudix motif," which is distribut...

The intestinal microbiome produces various metabolites that may harm or benefit the host. However, the production pathways of these metabolites have not been well characterised. The polyamines putrescine and spermidine required for physiological process are also produced by intestinal microbiome. The production and release of these polyamines by microbiome are poorly understood, though we have confirmed that intestinal bacteria produced putrescine from arginine. In this study, we characterised polyamine synthesis by analysing the collective metabolic functions of the intestinal microbiome. In particular, we analysed polyamines and their intermediates in faecal cultures, as well as the colonic contents of rats injected with isotope-labelled arginine through a colon catheter, using mass spectrometry. Isotope-labelled putrescine was detected in faecal cultures and colonic contents of rats injected with isotope-labelled arginine. Putrescine is produced through multiple pathways, and its extracellular intermediates are exchanged between bacterial species. Additionally, we demonstrated that the collective metabolic pathway depends on a complex exchange of metabolites released into the colonic lumen. This study demonstrates the existence of putrescine biosynthetic pathways based on the collective metabolic functions of the intestinal microbial community. Our findings provide knowledge to manipulate the levels of intestinal microbial products, including polyamines, that may modulate host health.

Low-molecular-weight metabolites produced by the intestinal microbiome play a direct role in health and disease. However, little is known about the ability of the colon to absorb these metabolites. It is also unclear whether these metabolites are bioavailable. Here, metabolomics techniques (capillary electrophoresis with time-of-flight mass spectrometry, CE-TOFMS), germ-free (GF) mice, and colonized (Ex-GF) mice were used to identify the colonic luminal metabolites transported to colonic tissue and/or blood. We focused on the differences in each metabolite between GF and Ex-GF mice to determine the identities of metabolites that are transported to the colon and/or blood. CE-TOFMS identified 170, 246, 166, and 193 metabolites in the colonic feces, colonic tissue, portal plasma, and cardiac plasma, respectively. We classified the metabolites according to the following influencing factors: (i) the membrane transport system of the colonocytes, (ii) metabolism during transcellular transport, and (iii) hepatic metabolism based on the similarity in the ratio of each metabolite between GF and Ex-GF mice and found 62 and 22 metabolites that appeared to be absorbed from the colonic lumen to colonocytes and blood, respectively. For example, 11 basic amino acids were transported to the systemic circulation from the colonic lumen. Furthermore, many low-molecular-weight metabolites influenced by the intestinal microbiome are bioavailable. The present study is the first to report the transportation of metabolites from the colonic lumen to colonocytes and somatic blood in vivo, and the present findings are critical for clarifying host-intestinal bacterial interactions.