The ATP-hydrolyzing ectoenzyme E-NTPD8 attenuates colitis through modulation of P2X4 receptor-dependent metabolism in myeloid cells.

Extracellular adenosine triphosphate (ATP) released by mucosal immune cells and by microbiota in the intestinal lumen elicits diverse immune responses that mediate the intestinal homeostasis via P2 purinergic receptors, while overactivation of ATP signaling leads to mucosal immune system disruption, which leads to pathogenesis of intestinal inflammation. In the small intestine, hydrolysis of luminal ATP by ectonucleoside triphosphate diphosphohydrolase (E-NTPD)7 in epithelial cells is essential for control of the number of T helper 17 (Th17) cells. However, the molecular mechanism by which microbiota-derived ATP in the colon is regulated remains poorly understood. Here, we show that E-NTPD8 is highly expressed in large-intestinal epithelial cells and hydrolyzes microbiota-derived luminal ATP. Compared with wild-type mice, Entpd8-/- mice develop more severe dextran sodium sulfate-induced colitis, which can be ameliorated by either the depletion of neutrophils and monocytes by injecting with anti-Gr-1 antibody or the introduction of P2rx4 deficiency into hematopoietic cells. An increased level of luminal ATP in the colon of Entpd8-/- mice promotes glycolysis in neutrophils through P2x4 receptor-dependent Ca2+ influx, which is linked to prolonged survival and elevated reactive oxygen species production in these cells. Thus, E-NTPD8 limits intestinal inflammation by controlling metabolic alteration toward glycolysis via the P2X4 receptor in myeloid cells.

Evaluation of change in metabolome caused by comprehensive diabetes treatment: A prospective observational study of diabetes inpatients with gas chromatography/mass spectrometry-based non-target metabolomic analysis.

AIMS/INTRODUCTION: Diabetes patients develop a variety of metabolic abnormalities in addition to hyperglycemia. However, details regarding change in various metabolites after comprehensive diabetes treatment remain unknown. This study aimed to identify the short-term change in metabolome in inpatients who were subject to comprehensive diabetes treatment, using gas chromatography/mass spectrometry-based non-target metabolomics techniques. MATERIALS AND METHODS: Participants of the present study were randomly recruited from the patients with type 2 diabetes hospitalized due to problems with glycemic control (n = 31) and volunteers without diabetes (n = 30), both of whom were aged between 20 and 75 years. A metabolomic analysis of fasting plasma samples on the 2nd (pre-treatment) and 16th hospital (post-treatment) day with gas chromatography/mass spectrometry using a multiple reaction monitoring mode was carried out. RESULTS: A principal component analysis showed that metabolome of fasting plasma was different between individuals with and without diabetes. The metabolome of fasting plasma in diabetes patients after treatment was different from that of pre-treatment, as well as individuals without diabetes. Many amino acids (proline, glycine, serine, threonine, methionine, pyroglutamic acid, glutamine and lysine) were significantly increased by >10% after administering the inpatient diabetes treatment. A hierarchical clustering analysis showed that in the case of patients with markedly decreased monosaccharide levels and increased 1,5-anhydroglucitol, the levels of amino acids increased more significantly. CONCLUSIONS: After a 2-week comprehensive treatment, the plasma levels of various amino acids increased in conjunction with the reduction in monosaccharide levels in poorly controlled type 2 diabetes patients.

Minimization of adverse effects of blank matrices from various apparatuses in the downsizing of gas chromatography-mass spectrometry-based metabolomics.

The ability to reduce sample volume required for gas chromatography-mass spectrometry (GC/MS) metabolome analyses is limited by the effects of blank matrices. In this study, a GC/MS metabolome analytical method requiring only 5 µL of plasma obtained by fingertip puncture, was developed, while minimizing the adverse effects of blank matrices. The applicability of the newly developed method was investigated using mice tail venous blood. Removing the effects of blank matrices greatly affected the detection repeatability for trace amounts. The newly developed method has higher metabolite coverage and higher sensitivity than those of the conventional method. This study is the first to demonstrate that comparable, or improved, metabolome profile data can be obtained with one-tenth the plasma volume required for the conventional method.

Plasma metabolites associated with arterial stiffness in patients with type 2 diabetes.

BACKGROUND: Although an increased arterial stiffness has been associated with traditional coronary risk factors, the risk factors and pathology of arterial stiffness remain unclear. In this study, we aimed to identify the plasma metabolites associated with arterial stiffness in patients with type 2 diabetes mellitus. METHODS: We used the metabolomic data of 209 patients with type 2 diabetes as the first dataset for screening. To form the second dataset for validation, we enlisted an additional 31 individuals with type 2 diabetes. The non-targeted metabolome analysis of fasting plasma samples using gas chromatography coupled with mass spectrometry and the measurement of brachial-ankle pulse wave velocity (baPWV) were performed. RESULTS: A total of 65 annotated metabolites were detected. In the screening dataset, there were statistically significant associations between the baPWV and plasma levels of indoxyl sulfate (r = 0.226, p = 0.001), mannitol (r = 0.178, p = 0.010), mesoerythritol (r = 0.234, p = 0.001), and pyroglutamic acid (r = 0.182, p = 0.008). Multivariate regression analyses revealed that the plasma levels of mesoerythritol were significantly (ß = 0.163, p = 0.025) and that of indoxyl sulfate were marginally (ß = 0.124, p = 0.076) associated with baPWV, even after adjusting for traditional coronary risk factors. In the independent validation dataset, there was a statistically significant association between the baPWV and plasma levels of indoxyl sulfate (r = 0.430, p = 0.016). However, significant associations between the baPWV and plasma levels of the other three metabolites were not confirmed. CONCLUSIONS/INTERPRETATION: The plasma levels of indoxyl sulfate were associated with arterial stiffness in Japanese patients with type 2 diabetes. Although the plasma levels of mannitol, mesoerythritol, and pyroglutamic acid were also associated with arterial stiffness, further investigation is needed to verify the results.

Identification of Plasma Inositol and Indoxyl Sulfate as Novel Biomarker Candidates for Atherosclerosis in Patients with Type 2 Diabetes. -Findings from Metabolome Analysis Using GC/MS.

AIM: An identification of the high-risk group of atherosclerotic cardiovascular disease (CVD) is important in the management of patients with diabetes. Metabolomics is a potential tool for the discovery of new biomarkers. With this background, we aimed to identify metabolites associated with atherosclerosis in patients with type 2 diabetes mellitus (T2DM). METHODS: A total of 176 patients with T2DM who have never had a CVD event and 40 who were survivors of coronary artery disease (CAD) events were enrolled. Non-targeted metabolome analysis of fasting plasma samples was performed using gas chromatography coupled with mass spectrometry (GC/MS) highly optimized for multiple measurement of blood samples. First, metabolites were screened by analyzing the association with the established markers of subclinical atherosclerosis (i.e., carotid maximal intima-media thickness (max-IMT) and flow-mediated vasodilation (FMD)) in the non-CVD subjects. Then, the associations between the metabolites detected and the history of CAD were investigated. RESULT: A total of 65 annotated metabolites were detected. Non-parametric univariate analysis identified inositol and indoxyl sulfate as significantly (p＜0.05) associated with both max-IMT and FMD. These metabolites were also significantly associated with CAD. Moreover, inositol remained to be associated with CAD even after adjustments for traditional coronary risk factors. CONCLUSIONS: We identified novel biomarker candidates for atherosclerosis in Japanese patients with T2DM using GC/MS-based non-targeted metabolomics.