It is crucial that the host and intestinal microflora interact and influence each other to maintain homeostasis and trigger pathological processes. Recent studies have shown that transplantation of the murine intestinal content to recipient germ-free mice enables transmission of the donor's phenotypes, such as low level chronic inflammation associated with lifestyle-related diseases. These findings indicate that intestinal bacteria produce some molecules to trigger pathological signals. However, fecal microbial metabolites that induce obesity and the type II diabetic phenotype have not been fully clarified. Here, we showed that the intestinal bacterial metabolite stercobilin, a pigment of feces, induced proinflammatory activities including TNF-α and IL-1β induction in mouse macrophage RAW264 cells. Proinflammatory stercobilin levels were significantly higher in ob/ob mice feces than in the feces of control C57BL/6 J mice. Moreover, in this study, we detected stercobilin in mice plasma for the first time, and the levels were higher in ob/ob mice than that of C57BL/6 J mice. Therefore, stercobilin is potentially reabsorbed, circulated through the blood system, and contributes to low level chronic inflammation in ob/ob mice. Since, stercobilin is a bioactive metabolite, it could be a potentially promising biomarker for diagnosis. Further analyses to elucidate the metabolic rate and the reabsorption mechanism of stercobilin may provide possible therapeutic and preventive targets.www.nature.com/scientificreports www.nature.com/scientificreports/ NF-κB-RE vector (Promega) using X-tremeGENE HP DNA Transfection Reagent (Roche). Luciferase activities were evaluated using the Nano-Glo Luciferase assay kit.Bligh-dyer extraction of feces. Fecal extracts were prepared by the Bligh-Dyer extraction method.Briefly, 30 mg feces suspended in a tube containing 0.5 mg of tert-butylhydroquinone (tBHQ) and 650 µL of MeOH/CHCl 3 /H 2 O = 400/200/40 was microdestructed (3000 rpm, 4°C, 120 s ×2 sets) by MicroSmash MS-100R (TOMY, Japan) with a 5.0 ϕ zirconia (ZrO 2 ) bead. After remove the ZrO 2 bead, the tube was centrifuged, and the aqueous and organic phases were harvested in new tubes. Soluble metabolites were extracted again by adding 200 µL of MeOH/CHCl 3 = 100/100 to the residue, vortexed, centrifuged, combined with former ones, then dried by a centrifuge evaporator.Lc-MS. Fecal metabolites were analyzed using LC-MS consisting of AQUITY UPLC (Waters, Milford, MA) coupled with micrOTOFQII (Bruker Daltonics, Bremen, Germany). UPLC separation 39,40 was performed with a CSH C18 column (1.7 μm, 100-mm × 2.1-mm i.d., Waters) at 40°C, using solvent A (0.1% formic acid in water) and solvent B (MeCN containing 0.1% formic acid). Samples were eluted from the column using a linear gradient of 1% solvent B from 0 to 3 min to 80% solvent B at 20-24 min. The flow rate of the mobile phase was 0.4 ml/min. The TOF-MS was operated in positive and negative ion mode using an electrospray ionization source. The detector conditions were as follows: capi...

When the microfloral composition deteriorates, it triggers low-level chronic inflammation associated with several lifestyle-related diseases including obesity and diabetic mellitus. Fecal volatile organic compounds (VOCs) have been found to differ in gastrointestinal diseases as well as intestinal infection. In this study, to evaluate a potential association between the pathogenesis of lifestyle-related diseases and VOCs in the intestinal tract, fecal VOCs from obese/diabetic KK-A y mice (KK) or controls (C57BL/6J mice; BL) fed a normal or high fat diet (NFD or HFD) were investigated using headspace sampler-GC-EI-MS. Principal component analysis (PCA) of fecal VOC profiles clearly separated the experimental groups depending on the mouse lineage (KK vs BL) and the diet type (NFD vs HFD). 16 s rRNA sequencing revealed that the PCA distribution of VOCs was in parallel with the microfloral composition. We identified that some volatile metabolites including n-alkanals (nonanal and octanal), acetone and phenol were significantly increased in the HFD and/or KK groups. Additionally, these volatile metabolites induced proinflammatory activity in the RAW264 murine macrophage cell line indicating these bioactive metabolites might trigger low-level chronic inflammation. These results suggest that proinflammatory VOCs detected in HFD-fed and/or diabetic model mice might be novel noninvasive diagnosis biomarkers for diabetes.

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