Animals
Forty-one male C57BLKS/Jlar-+Lepr/db+Lepr/db (db⁄db) mice aged 6 weeks were purchased from Japan SLC, Inc. (Shizuoka, Japan). They were maintained under controlled temperature (23 ±2 °C) and light-dark cycle with free access to food and water, and fed a regular chow diet (5.1% fat, 55.3% carbohydrate, 23.1% protein; MF Oriental Yeast Co., Ltd., Tokyo, Japan). After acclimatization for a week, the mice were randomly assigned to Pg-treated (n = 20) and CMC-treated (n = 21) groups. The bacterial load administered in the mouse periodontitis model was based on Baker et al. 32. Pg and CMC were administered orally through a plastic tube, with 109 CFU Pg mixed with 4% CMC () (for the Pg-treatment), or only CMC (for the control), every 3 d for 30 d. Livers were excised after anesthetization using mixed anesthesia (Domitor, 0.75 mg/kg body weight; Midazolam, 4 mg/kg; and Butorphanol Tartrate, 5 mg/kg), and were flash-frozen in liquid nitrogen.
The oral glucose tolerance test was performed following overnight (10 h) fasting. Fasting glucose levels were measured, and mice were orally administered with 2 g glucose/kg body weight. The intraperitoneal insulin tolerance test was conducted by intraperitoneal insulin injections (5 units/kg body weight). Blood glucose levels were measured at 0, 30, 60, and 120 min after insulin administration.
To determine insulin levels, blood samples were collected from the inferior vena cava of anesthetized mice. Serum insulin levels were determined using the insulin ELISA kit (FujiFilm Wako Shibayagi Corporation, Gunma, Japan), following the manufacturer’s instructions.
All animal experiments were performed according to the protocols approved by the institutional animal care and use committees of Osaka University Graduate School of Dentistry (permit number: 27-022-0) and were in accordance with the Guide for the Care and Use of Laboratory Animals, published by the National Academies Press. All mice were manipulated in accordance with Animal Research Reporting In Vivo Experiments (ARRIVE) guidelines.
Bacterial culture
The Pg strain (ATCC33277) was obtained from the American Type Culture Collection (ATCC, Manassas, VA) and grown at 37 °C for 24 h in an anaerobic box chamber (Mitsubishi Gas Chemical Company, Inc. Tokyo, Japan) with AnaeroPack-Anaero anaerobic gas generator (Mitsubishi Gas Chemical Company, Inc.) in Gifu anaerobic medium supplemented with 5 mg/mL yeast extract, 5 μg/mL hemin, and 0.2 μg/mL vitamin K1.
Quantification of alveolar bone resorption
Morphometric analysis of buccal alveolar bone resorption was performed using an R_mCT2 3D micro X-ray computed tomography system designed for use with scanned images of laboratory animals (Rigaku, Tokyo, Japan). An examiner blinded to the experimental groups measured the linear distances of the cementoenamel junction (CEJ) from the alveolar bone crest (ABC) using the 3D image analysis software TRI/3D-BON (RATOC System Engineering Co., Ltd., Tokyo, Japan). Five linear points were measured on each molar root surface (three on the distal root, two on the mesial root).
Real-time PCR
Total RNA from the mouse liver was extracted using an RNeasy lipid tissue mini kit (Qiagen, Venlo, Netherlands) according to the manufacturer’s instructions. cDNA was synthesized from 100 ng total RNA using a high-capacity cDNA archive kit (Applied Biosystems, Foster City, CA). PCR was performed using the ABI 7300 real-time PCR system with the Power SYBR Green PCR master mix (both from Applied Biosystems) according to the manufacturer’s protocol. To control for variation in the amount of DNA available for PCR, target gene expression in each sample was standardized based on expression of an endogenous control. The sequences of the primers used are provided in Supplementary Table S3.
Protein analysis
Total proteins were extracted from the frozen livers using the T-PER tissue protein extraction reagent (Thermo Fisher Scientific Inc., Waltham, MA), and used for western blotting. Immunoblotting was performed using the following primary antibodies: PCK1 (ab28455, Abcam, Toronto, Canada), G6PC (ab83690; Abcam), FOXO1 (2880, Cell Signaling Technology, Danvers, MA), and b-actin (A5216, Sigma‐Aldrich Co., St. Louis, MO).
Histology
Liver tissues, excised from mice after the 30 days period of oral administration of Pg- and CMC-treatment, were fixed using 4% paraformaldehyde for 48 h and embedded in paraffin. The tissue sections were then deparaffinized, rehydrated, and washed with PBS. The tissue sections were cut at 4 μm and stained with hematoxylin and eosin (H&E). For immunohistochemistry, samples were embedded in paraffin, sectioned, and stained with rabbit anti-PCK1 (ab2845, 0.4 µg/mL; Abcam) and rabbit anti-FOXO1 antibodies (2880, 0.2 µg/mL; Cell Signaling Technology). Positive staining was visualized using a Simple Stain DAB Solution.
Metabolome analysis
Metabolites were extracted from the frozen small intestines or frozen livers using Bligh and Dyer’s method 33. Metabolome analysis of the small intestines was performed at the Chemicals Evaluation and Research Institute (CERI, Saitama, Japan) via gas chromatography triple quadrupole mass spectrometry (GC/MS/MS) and ion-paring liquid chromatography triple quadrupole mass spectrometry (ion-pairing LC/MS/MS) 34. Hydrophilic metabolites of the liver were analyzed using ion chromatography coupled with a high-resolution tandem mass spectrometer (IC/MS/MS) for anionic polar metabolites such as organic acids and nucleotides 35, and liquid chromatography with a pentafluorophenyl propyl column coupled with a high-resolution tandem mass spectrometer (PFPP-LC/MS/MS) for cationic polar metabolites such as amino acids 35. The free fatty acid (FA) and cholesteryl ester (ChE) lipid content of the livers was quantified using supercritical fluid chromatography with a C18 column coupled with triple quadrupole mass spectrometry (C18-SFC/MS/MS) 36. The levels of other lipids—phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidic acid (PA), lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), monoacylglycerol (MG), diacylglycerol (DG), triacylglycerol (TG), sphingomyelin (SM), cholesterol, ceramide (Cer), and hexosylceramide (HexCer)—were quantified using SFC with a diethylamine (DEA) column coupled with triple quadrupole mass spectrometry (DEA-SFC/MS/MS) 37. Details regarding sample preparation and the analytical conditions for hydrophilic and hydrophobic metabolite analysis are provided as Supplementary Methods.
Determination of liver glycogen content
The glycogen content of liver tissue was determined using an aqueous size-exclusion chromatographic method previously reported 38.
Proteome analysis of liver enzymes and gut microbiota in fecal specimens
Liver and fecal samples of the 12-week-old male db/db mice After the 30 days treatment were collected and cut into small pieces with dissection scissors. For fecal samples, 450 µL methanol was added to 10 mg feces, and then 90 µL of the suspension was diluted with 450 µL methanol. Distilled water (250 µL) and 500 µL chloroform were added to the diluted suspension, followed by vortexing. After centrifugation at 4,600 ´g for 5 min, both the organic and aqueous phases were removed, and pellets in the interphase were dried under vacuum. Proteins were extracted from the dried extract of the feces and the disrupted livers using the phase-transfer surfactant method 39 with a slight modification. The extracted protein was subjected to reductive alkylation, followed by successive digestion with Lys-C endopeptidase and trypsin, as previously described 40.
The mouse liver digests were isotopically labelled with TMT 10-plex 41 (Thermo Fisher Scientific) according to the manufacturer’s protocol. For parallel reaction monitoring (PRM) analysis, the digests of fecal samples and of the synthetic peptide were isotopically labelled via reductive dimethylation 42. The digested peptides were analyzed using nano-LC/MS/MS, using an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific) in data-dependent acquisition (DDA) mode, or using a Q Exactive mass spectrometer (Thermo Fisher Scientific) in PRM mode, coupled to Ultimate3000 RSLCnano system (CTC Analytics) and HTC-PAL autosampler (CTC). Details regarding sample preparation, the analytical conditions for nano-LC/MS/MS, and data processing, are described in the Supplementary Methods.
Statistical analysis
All data are presented as mean ± SEM. Differences in body weight, food intake, and blood glucose level between the Pg and CMC (control) groups were analyzed using one-way ANOVA with Tukey’s post hoc test. All other comparisons between the two groups were analyzed using an unpaired t-test. Differences were considered statistically significant at P < 0.05.