Adenosine

SVF cells containing ADSCs were isolated from epididymal and inguinal adipose tissue according to our previous study [8 (link),9 (link)]. In brief, adipose tissues were minced with scissors and placed in plastic vials in the isolation buffer (Krebs–Ringer bicarbonate solution buffered with 10 mM HEPES, pH 7.4, containing 5.5 mM glucose and 2.5% (w/v) fatty acid-free bovine serum albumin) with 200 nM adenosine and collagenase type 1 (2 mg/mL; Wako Pure Chemical Industries, Ltd., Osaka, Japan). Collagenase digestion was performed at 37 °C in a water-bath shaker. After 45 min, the vials were filtered and centrifuged at 600× g for 10 min. SVF pellets were then washed twice with phosphate-buffered saline and seeded onto a 10-cm dish in Dulbecco’s modified Eagle medium (DMEM; Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Gibco, Grand Island, NY, USA), and maintained at 37 °C in a 5% CO₂ atmosphere. At 80% confluence, the cultures were passaged using 0.25% Trypsin/EDTA (Gibco) and reseeded at a density of 1.0 × 10⁴ cells/cm². SVF cells between passages three to four were used for subsequent experiments as ADSCs. Cultured SVF cells appeared as fibroblast-like cells and acquired a homogenous spindle shape after three passages, and their features were consistent with isolated ADSCs [75 (link),76 (link),77 (link)].

T. b. brucei bloodstream forms of strain GUTat 3.1 and TC221 maintained at Tokyo Medical and Dental University were used for all in vitro and in vivo experimental procedures. Parasites were cultured in Iscove’s modified Dulbecco’s medium (IMDM) (Sigma-Aldrich, St. Louis, MO, USA) supplemented with two mixtures; mixture 1 consisted of 0.1 M HCl, 100 μM hypoxanthine (Sigma-Aldrich), 30 μM thymidine (Sigma-Aldrich), and 40 μM adenosine (WAKO, Osaka, Japan) and mixture 2 consisted of a mixture of 1 mM sodium pyruvate (WAKO), 200 μM l-alanine (WAKO), 100 μM glycine (WAKO), 20 μM l-ornithine monohydrochloride (Tokyo Kasei, Tokyo, Japan), 10 μM l-citrullin (Sigma-Aldrich) and 100 μM 2-mercaptoethanol (WAKO), and 200 ml of distilled water. The adjusted IMDM had a further 10 % fetal bovine serum (FBS), 2 mM l-glutamine (WAKO), and 100 U/ml penicillin-100 μg/ml streptomycin added to it. Parasites were cultured at 37 °C and 5 % CO₂ humid atmosphere as described previously [12 (link)].

B. breve MCC1274 was cultured in YC medium supplemented with 200 μM each of adenosine, inosine, hypoxanthine, and xanthine (FUJIFILM Wako Pure Chemical) for 24 h under anaerobic conditions. Then, the culture medium was centrifuged (10,000 × g, 5 min, 4°C), and the supernatant was stored at −20°C until analysis.

Mice were divided into groups based on glucose levels in whole blood collected from the tail vein using LIFE CHECK (EIDIA Co., Ltd., Tokyo, Japan). The mice were subcutaneously injected with adenosine (Wako Pure Chemicals Industries, Ltd., Osaka, Japan) dissolved in phosphate-buffered saline (PBS). The same volume of PBS was injected as a control. Post injection, glucose levels in whole blood were measured from the tail vein. The area under the curve (AUC) was calculated from glucose levels during the experiment.