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.38. 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 days for 30 days. The experiment was divided into four administrating sessions with 4–6 animals in each group. The food intake in db/db mice was measured for 6–11 consecutive weeks. The animals and the amount of food in the cage were weighed once a week. After anesthetization using mixed anesthesia (Domitor, 0.75 mg/kg body weight; Midazolam, 4 mg/kg; and Butorphanol Tartrate, 5 mg/kg), blood was collected from inferior vena cava and liver samples were excised and harvested for the following experiments. Maxillae were removed from euthanized db⁄db mice and fixed using 4% paraformaldehyde for 48 h.
The oral glucose tolerance test was performed following overnight (10 h) fasting, 21 days after the initial Pg treatment. Fasting glucose levels were measured, and mice were orally administered with 2 g glucose/kg body weight. The intraperitoneal insulin tolerance test was conducted with intraperitoneal insulin injections (5 units/kg body weight), 26 days after the initial Pg treatment. 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). In addition, all methods were performed in compliance with the 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 the 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 cemento-enamel junction (CEJ) from the alveolar bone crest (ABC) using the 3D image analysis software TRI/3D-BON (RATOC System Engineering Co., Ltd., Tokyo, Japan). Buccal-side maxillary alveolar bone loss (ABL) was measured from the cemento-enamel junction (CEJ) to alveolar bone crest (ABC) at five points: (1) distobuccal regions for first maxillary molar (M1); (2) mesiobuccal and (3) distobuccal regions for second maxillary molar (M2); and (4) mesiobuccal and (5) distobuccal regions for third maxillary molar (M3), after 30 days following the treatment with Pg or CMC in db/db mice. Distance between the CEJ and the ABL was measured at five sites in the apical direction using WinROOF software version 7.4 (https://www.mitani-visual.jp/products/#image_analys_ismeasurement) (Mitani Corporation, Fukui, Japan), and total value of five points on the μCT image was defined as the alveolar bone loss, were compared in Pg or CMC-control treated groups. Prior to the observation, the intraclass correlation for the evaluation of bone loss measurements was examined. One examiner evaluated the same teeth points on different days. The resulting intraclass correlation coefficient was 0.86.
Real-time PCR
Total RNA from the mouse liver was extracted using a 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 the variations in the amount of DNA available for PCR, target gene expression in each sample was standardized based on the expression of an endogenous control. The sequences of the primers used are provided in Supplementary Table S2.
Protein analysis
Total proteins were extracted from the frozen liver tissues 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 (1:1000; ab28455, Abcam, Toronto, Canada), G6PC (ab83690; Abcam), FOXO1 (1:000; 2880, Cell Signaling Technology, Danvers, MA), and β-actin (A5216, Sigma‐Aldrich Co., St. Louis, MO), and incubated with anti- rabbit HRP-conjugated secondary antibody (1:10,000; NA934, GE Healthcare, Chicago, IL, USA) anti- mouse HRP (1:10,000; NA931, GE Healthcare). Immunoreactive bands were visualized using ECL (Thermo Fisher Scientific).
Histology
Liver tissues, excised from mice after 30 days following the oral administration of Pg and CMC, were fixed using 4% paraformaldehyde for 48 h and embedded in paraffin. Samples were then deparaffinized, rehydrated, and washed with PBS. The tissue sections were cut at 4 μm thickness with LEICA RM2245 (Leica Microsystems, Wetzlar, Germany) 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 diaminobenzidine (DAB) in stable peroxide buffer.
Metabolome analysis
Metabolites were extracted from the frozen small intestine or frozen liver samples using Bligh and Dyer’s method39. Metabolome analysis of the small intestines was performed at the Chemicals Evaluation and Research Institute (CERI, Saitama, Japan) using gas chromatography triple quadrupole mass spectrometry (GC/MS/MS) and ion-paring liquid chromatography triple quadrupole mass spectrometry (ion-pairing LC/MS/MS)40. The 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 nucleotides41; and with 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 acids41. The levels of free fatty acids (FAs) and cholesteryl esters (ChEs) in the liver samples were quantified using supercritical fluid chromatography with a C18 column coupled with triple quadrupole mass spectrometry (C18-SFC/MS/MS)42. 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)43. Details regarding sample preparation and the analytical conditions for the analysis of the hydrophilic and hydrophobic metabolites are provided as Supplementary Methods.
Determination of liver glycogen content
The glycogen content in liver tissues was determined using an aqueous size-exclusion chromatographic method, as reported previously44.
Proteome analysis of liver samples and gut microbiota in fecal specimens
Liver and fecal samples of the 12-week-old male db/db mice were collected 30 days after the treatment and cut into small pieces using dissection scissors. For fecal samples, 450 µL methanol was added to 10 mg feces, and 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 vertexing. After centrifugation at 4600×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 method45, with a slight modification. The extracted protein was subjected to reductive alkylation, followed by successive digestion with Lys-C endopeptidase and trypsin, as previously described46.
The mouse liver digests were isotopically labelled with TMT 10-plex47 (Thermo Fisher Scientific), according to the manufacturer’s protocol. For PRM analysis, the digests of fecal samples and that of the synthetic peptide were isotopically labelled via reductive dimethylation48. 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 RSLC nano system (CTC Analytics) and the 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 levels 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.
