Animals
Male C57BL/6J mice were housed in cages in a temperature-controlled room under a 12-h light-dark cycle. During a 1-wk adaptation period, all mice were fed standard chow and water ad libitum. After an initial acclimatization period, animals (2 mo of age) were randomly assigned to the following groups: 4-mo low-fat diet (LFD; n = 6), 4-mo HFD (n = 6), 20-mo LFD (n = 6), and 20-mo HFD (n = 5). twenty-one animals investigated herein represent a subset of mice from a larger cohort in 10 which data related to the assessment of the morphology have not been reported. The HFD mice were fed a diet containing 20% protein, 60% fat, and 20% carbohydrate (D12492; Research Diets, New Brunswick, NJ), and the LFD mice were fed standard chow containing 22.6% protein, 5.6% fat, and 53.8% carbohydrate [a pelleted Charles River Formula (CRF)-1 diet; Charles River, Japan]. After 4 mo (when mice were 6 mo old) or 20 mo (when mice were 22 mo old) of the HFD or standard chow diet, Mice were anesthetized via intraperitoneal injection of sodium pentobarbital (70 mg/kg body wt), and their muscles were dissected once a surgical level of anesthesia was reached.
Histology
Mouse muscle fibers in the histological sections were examined and analyzed as described previously 11. Briefly, serial 10-μm sections were cut with a cryostat (CM1510; Leica, Tokyo, Japan) at −20°C and mounted on polylysine-coated slides. Whole sections were stained for hematoxylin-and-eosin (H&E), slow and fast myosin heavy chains (MHCs). Mouse monoclonal antibodies that react specifically with type I (BF-F3), type IIa (SC-71), or type IIx (BF-35) MHC isoforms were supplied by the Developmental Studies Hybridoma Bank (University of Iowa, IA). The M.O.M. Immunodetection kit (Vector Laboratories, Burlingame, CA) and Vectastain ABC kit (Vector Laboratories) were used to assess immunohistochemical reactions according to the manufacturer’s instructions. Images were recorded using a camera (E1000M; Nikon, Japan) at ×10 magnification and analyzed subsequently in ImageJ (NIH, Bethesda, MD). The cross-sectional areas (CSA) and Feret’s diameter were measured by tracing fiber outlines of ∼371 fibers from the muscle sections of any individual mouse.
Statistical analysis
Values are expressed as means ± SE. Statistical analyses were performed with Prism version 9.0 (GraphPad Software, San Diego, CA). For comparisons of groups, two-way ANOVA with a Tukey’s post-hoc test was used. For all tests, P < 0.05 was considered statistically significant.
Approval for animal experiments
All animal experiments in this study were approved by the Animal Experimental Committee of Juntendo University. All experimental animals were handled according to institutional guidelines for the care and use of laboratory animals. This manuscript was prepared according to the Animal Research: Reporting of In Vivo Experiments guidelines (https://arriveguidelines.org).