Male C57BL/6J mice were purchased from Japan SLC (Shizuoka, Japan) and maintained under a strict 12-h light/dark cycle in a conventional animal room at 23.0 °C and 40-70% relative humidity. The mice were acclimated to the laboratory conditions on the CLEA Rodent Diet (CE-2, CLEA Japan, Inc., Tokyo, Japan) for 1 week prior to the treatment. 12 per group). The compositions of the diets are given in Supplementary Table S1. These diets were adjusted so that the final percentages of protein, fat, and carbohydrates were almost equal in all groups. 8-10 per group). The generation of Ffar3−/− and Ffar2−/− mice was described previously36,40. The body weights were measured once a week. Food intake was calculated as the average of daily food intake (g/day per mouse) for 4 weeks. All mice were then sacrificed under deep isoflurane-induced anaesthesia, and the liver, caecum, epididymal, perirenal, and subcutaneous adipose tissues were harvested and weighed. Blood was collected from the inferior vena cava using heparinised tubes and plasma was separated by immediate centrifugation (7,000 × g, 5 min, 4 °C).
For intraperitoneal phosphate buffered saline (for control group) or propionate administration (sodium propionate, Wako, 1 g/kg body weight) as previous described36, 7-week-old wild-type and Ffar3−/− mice on C57BL/6J background were sacrificed and collected samples at 24 h after propionate administration under HFD feeding. All tissues and plasma were stored at −80 °C until further processing. All experimental procedures involving mice were performed in 2017-2019 according to protocols approved by the Committee on the Ethics of Animal Experiments of the Tokyo University of Agriculture and Technology (Permit No. 28-87). All efforts were made to minimise suffering. The blood glucose concentration was measured using One Touch Ultra Test Strips (One Touch® Ultra®, Life Scan, Milpitas, CA, USA). The concentrations of plasma total cholesterol (Lab Assay™ Cholesterol, Wako, Tokyo, Japan), NEFAs (Lab Assay™ NEFA, Wako, Tokyo, Japan), plasma and hepatic TG (Lab Assay™ Triglyceride, Wako, Tokyo, Japan), plasma PYY (Mouse/Rat PYY ELISA Kit, Wako, Tokyo, Japan), GLP-1 (GLP-1 Active ELISA Kit, Merck Millipore, Darmstadt, Germany), and insulin (Insulin ELISA KIT (RTU), Shibayagi, Gunma, Japan) were measured following the manufacturer’s instructions.
For plasma GLP-1 measurement, the plasma sample was treated with a dipeptidyl peptidase IV inhibitor (Merck Millipore, Darmstadt, Germany) to prevent the degradation of active Medic GLP Website-1. SCFA levels in the plasma and caecum were determined following a modification of a previously described protocol49. Herein, the SCFA-containing ether layers were collected and pooled for gas chromatography-mass spectrometry analysis using a GCMS-QP2010 Ultra system (Shimadzu, Kyoto, Japan). The calibration curves for SCFAs were constructed, and the concentration of each SCFA in the samples was evaluated over a specified concentration range. For GTT, 16-h fasted mice were given 2.0 mg of glucose per gram of body weight intraperitoneally (i.p.). For ITT, 3-h fasted mice were given human insulin (0.75mUg−1, i.p., Sigma). The blood glucose concentration was measured before injection and monitored at 15, 30, 60, 90 and 120 min after injection. The liver contents were weighed and stored at −80 °C until further processing. Hepatic triacylglycerol contents were measured following a modification of a previously described protocol40.
Briefly, liver homogenates were subjected to crude lipid extraction using a mixture of chloroform/methanol/0.45 M acetic acid. Subsequently, Medic GLP Website 3 volumes of mixture were added and shaken overnight (4 °C). After centrifugation at 1,500 × g for 10 min, the organic layer was collected, dried, and resuspended in isopropyl alcohol. Measurements were conducted using the Lab Assay™ Triglyceride (Wako, Tokyo, Japan). Livers were embedded in OCT compounds (SAKURA, Finetek, Japan) and sectioned at 10 μm. Hematoxylin-eosin and oil red O (Sigma) staining were based on a previously described protocol with modification40, and examined with fluorescence microscope (All-in-One Fluorescence Microscope BZ-X700, KEYENCE, Osaka, Japan). Faecal DNA was extracted using Fast DNA® SPIN Kit (MP Biomedicals, Santa Ana, CA, USA), and the V4 region of the 16S rRNA gene was amplified using dual-indexed primers. The amplicons were then sequenced using an Illumina MiSeq with a MiSeq Reagent kit V3 (Illumina, San Diego, CA, USA).