Animal experiment
SAMP8 mice were used in this study. SAMP8 is a type of mouse whose age accelerates between 16 weeks and 20 weeks, and is often used in aging research. The mice were raised for each of the following groups: 12-week-old (young) (N=4) to a stage before the start of accelerated aging; 40-week-old (elderly) (N=4) in the mean lifespan of SAMP8; 55-week-old (late elderly) (N=5) to a stage that exceeded the mean lifespan. In this study, the same species and the same age as those used in the previous studies [8] were used. The mice were raised in a 125×213×125 mm aluminum cage and were freely given food (Lab MR-A1) and water. Upon administering general anesthesia with isoflurane, the mice were euthanized by dislocating the cervical spines, and the masseter muscles were extracted. The experiments were conducted in accordance with the National Institutes of Health guidelines for care and use of animals and also approved by the Tokyo Dental College Institutional Animal Care and Use Committee (approval number: 202601). The study was carried out in compliance with the ARRIVE guidelines.
Body weight and the amount of food intake
In this study, body weight was measured once a week with an electronic scale (AND EW-1500i) and the food was weighed concurrently with a balance from 12-week-old to 55-week-old. The following week, the feed was again measured concomitantly with the mice’s bodyweight, and the difference in the mass of the food between the two weeks represented the amount of food intake.
Capillary electrophoresis-mass spectrometry (CE-MS) metabolome analysis
Approximately 50 mg of frozen tissue was plunged into 750 µL of 50% acetonitrile/Milli-Q water containing internal standards (H33041002, Human Metabolome Technologies, Inc., Tsuruoka, Japan) at 0 °C to inactivate enzymes. The tissue was homogenized three times at 1,500 rpm for 2 min using a tissue homogenizer (Micro Smash MS100R, Tomy Digital Biology Co., Ltd., Tokyo, Japan) and then the homogenate was centrifuged at 2,300 ×g and 4 °C for 5 min. Subsequently, 400 µL of the upper aqueous layer was centrifugally filtered through a Millipore 5-kDa cutoff filter at 9,100 ×g at 4 °C for 120 min to remove proteins. The filtrate was centrifugally concentrated and re-suspended in 50 µL of Milli-Q water for capillary electrophoresis-mass spectrometry (CE-MS) analysis.
The metabolome analysis was performed with capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) for cation analysis and CE-tandem mass spectrometry (CE-MS/MS) for anion analysis based on the methods described previously. This analysis focused on 116 central metabolites. CE-TOFMS analysis was conducted using an Agilent CE capillary electrophoresis system equipped with an Agilent 6210 time-of-flight mass spectrometer (Agilent Technologies, Waldbronn, Germany).
Quantitative analysis of expression metabolites
Total RNA extraction of the masseter muscle was performed using the RNeasy Mini Kit (Qiagen, Hilden, Germany) and Proteinase K (Takara Bio, Shiga, Japan). The cDNA was prepared using the QuantiTect Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA). The Double Delta Ct Value (ΔΔCt) method was used for quantification, and the relative expression level was compared to the expression of housekeeping genes in each sample.
For gene expression analysis, the TaqMan® gene expression assays (Thermo Fisher Scientific, Massachusetts, USA) were used in the RT-PCR 7500 realtime polymerase chain reaction (PCR) system (Thermo Fisher Scientific). This study focused on the polyamine and purine metabolic pathways, which were the most common metabolites that fluctuated between 40-week-old and 55-week-old mice. For target genes in the polyamine metabolic pathway, TaqMan® probes
(Thermo Fisher Scientific) used Methionine Adenosyl Transferase 2A (Mat2a) (Mm00728688_s1), SRM (Mm00726089_s1), spermine synthase (SMS) (Mm00786246_s1), Ornithine decarboxylase1 (Odc1) (Mm02019269_g1), spermine oxidase (Smox) (Mm01275475_m1), and S-adenosylmethionine decarboxylase (Amd2) (Mm04207265_gH). Meanwhile, in the purine metabolic pathway, TaqMan® probes used HPRT (Mm03024075_m1), phosphoribosyl pyrophosphate 1 (Prps1) (Mm00727494_s1), adenine phosphoribosyl transferase (Mm04207855_g1), and xanthine dehydrogenase (Xdh) (Mm0044). In addition, the housekeeping gene β-actin (Mm00607939_s1) was used.
Immunohistochemical observations
The gathered masseter muscle was cut approximately 5 mm at the center of the belly of the muscle and fixed to a cork plate perpendicular to the muscle fibers. The specimens were rapidly frozen using isopentane cooled in liquid nitrogen. Frozen sections with a thickness of 4.5 μm and 20 μm were prepared using a cryostat M1950 (Leica, Nussloch, Germany), and then dried at room temperature for 60 min. In the immunohistochemical staining, the local expression of SRM in the polyamine metabolic pathway and HPRT in the purine metabolic pathway, which showed significant differences in gene expression, were observed in muscle fibers. The sections used for the SRM antibody were fixed with acetone for 10 min, and the sections used for the HPRT antibody were fixed with methanol for 10 min. After washing with PBS solution for 15 min, blocking was performed with 10% goat serum for 1 h and washed with PBS solution for 15 min. The primary antibody used was a 200fold diluted HPRT antibody (GeneTex, California, US) and SRM antibody (Proteintech, Chicago, US), and the specimen was incubated overnight at 4 °C after applying the primary antibody.
Furthermore, the secondary antibody, a goat anti-rabbit IgG antibody Alexa Fluor Plus 488 (Invitrogen, Massachusetts, US) was diluted 200-fold for the HPRT antibody and SRM antibody. To confirm the cytoskeleton, after applying the Acti-stain ™ 555 Fluorescent Phalloidin (Cytoskeleton Inc., Denver, US) secondary antibody, the sections were incubated at room temperature for 1 h under a shade. A universal microscope (Axioplan2 and Axiophot2, Carl Zeiss, Inc., Jena, Germany) and a confocal laser scanning microscope (LSM 880 with Airyscan, Carl Zeiss, Inc., Jena, Germany) were used for observation.
Statistical analyses
Statistical analyses were performed with IBM SPSS Statistics version 26 (IBM, Armonk, NY, USA) software. Moreover, for the statistics of body weight and food intake, Tukey's test was performed for the multiple comparison test. The metabolites, RT-PCR, and immunohistochemical stained areas of 40-week-old and 55-week-old mice were compared using Welch's t-test. The significance threshold was set at 5% (p < 0.05). The detection limit was described (p=0).