Table of Contents
All procedures were performed in accordance with the guidelines and regulations of the University of Miyazaki. This study was also conducted in accordance with his NIH Guidelines for Animal Care and Experimentation (ARRIVE). The research plan was approved by the Animal Experiment Ethics Review Committee, Faculty of Medicine, University of Miyazaki (approval number 2019-516).
Thirty-two male Wistar rats (7 weeks old, Charles River Japan, Shiga Prefecture) participated in this study. Rats were individually housed in plastic cages at constant room temperature under a 12:12 h light/dark cycle (lights on, 08:00–20:00). Rats were randomly assigned to two groups (n = 16 animals/group), CP (standard laboratory chow, 51% carbohydrate, 25% protein, 4.6% fat, CE-2, Nihon Clea, Tokyo, Japan) or SP (standard laboratory chow). Gave one. [1 kg] soaked in water [1.4 L]) 24 weeks. The hardness of CP exceeded 200 N, while that of SP was 0.32 N. The prepared SPs were stored at 4 °C and used within 1 week. Visible bacterial counts were similar between CP and SP, and no mold or yeast growth occurred. Hardness and bacterial testing were performed by the Japan Food Research Center (Tokyo, Japan). All rats were fed on a 3-h time-restricted feeding schedule from 10:00 to 13:00. All animals had free access to water and body weight and caloric intake were monitored weekly during the study period. Rats were deeply anesthetized with sodium pentobarbital (100 mg/kg body weight, ip) and euthanized by decapitation before collecting blood and tissues as a final procedure.
Insulin resistance (HOMA-IR) in rats was determined using the obtained fasting insulin and blood glucose measurements according to the following formula:17
Glucose tolerance tests were performed on rats fed CP or SP for 16 weeks (n= 10 pieces per group). Rats were fasted overnight. At 9:00 the next morning, each was injected intraperitoneally with glucose (2 g/kg body weight). To collect blood samples for measuring glucose and insulin levels, xylocaine 2% jelly (Lidocaine HCl, AstraZeneca, Osaka, Japan) was applied to the tail skin for topical anesthesia, and then the punctured tail was Blood was collected from a vein into a heparinized capillary tube. . Blood glucose levels were measured using a hand-held blood glucose meter (Breeze 2, Bayer, Osaka). The remaining sample was immediately centrifuged at ×1500.gAfter incubation at 4°C for 15 min, plasma was stored at -30°C until insulin assay. Plasma insulin levels were measured using an ELISA kit (Morinaga, Yokohama, Japan). Area under the curve (AUC) was calculated using Prism 8 (GraphPad Software, San Diego, CA, USA).
To measure triglyceride concentrations during feeding, plasma was prepared from blood collected from punctured tail veins into heparinized capillary tubes as previously described (n = 5 pieces per group). To analyze triacylglycerol content in liver and muscle, 25 mg of liver or 50 mg of skeletal muscle was homogenized using a BioMasher (Nippi, Tokyo, Japan) and then 500 ml of ice-cold chloroform:methanol. (2:1) was used to extract lipids. [v/v]) As mentioned above18. Triacylglycerol content of plasma, liver, and skeletal muscle samples was quantified using the LabAssay triglyceride kit (Wako Chemical, Tokyo, Japan).
Rats given CP or SP for 24 weeks were anesthetized (pentobarbital, 50 mg/kg body weight administered intraperitoneally) and subjected to micro-CT (LaTheta, Scanned at Aloka, Tokyo, Japan). Distal end of L6 (n= 10 pieces per group). Abdominal adiposity and lean body mass were then measured using LaTheta software (version 1.00).
Gastrocnemius and soleus muscle tissues from rats fed CP or SP for 24 weeks were cut at the mid-abdomen, frozen in liquid nitrogen, and stored at −80 °C. Continuous cross sections (thickness, 10 μm) were cut with a cryostat microtome. To measure CSA of individual fibers, cryostat sections of muscle were stained for laminin, a major component of the basal layer. For this purpose, sections were fixed in 10% neutral buffered paraformaldehyde and incubated in PBS containing 0.3% Triton X and 5% (v/v) normal goat serum (Thermo Fisher Scientific, Waltham, MA, USA) for 30 min. Transparent and blocked. room temperature. Sections were then incubated with primary antibody against laminin (dilution, 1:400, catalog number L9393, Sigma-Aldrich, St. Louis, MO, USA) overnight at 4 °C and incubated with Alexa Fluor 488. –conjugated goat anti-rabbit IgG (1:200; Thermo Fisher Scientific) for 40 min at room temperature. Stained sections were mounted with ProLong Gold (Thermo Fisher Scientific). The stained muscle samples of the CP and SP groups were imaged with a fluorescence microscope (All-in-One, Keyence, Osaka, Japan) and analyzed using the manufacturer-provided software (Keyensce).
Gastrocnemius and soleus skeletal muscles were collected from rats fed CP or SP for 24 weeks (n 30 min after intraperitoneal injection of saline or insulin (1 U/kg body weight; Eli Lilly Japan, Kobe, Japan) or after an overnight fast = 6 times per group). Muscle samples (25 mg each) were incubated in mammalian cell lysis reagent (ProteoJET, Fermentas Life Sciences, Ontario, Canada) containing proteinase inhibitor cocktail (Roche Diagnostics, Basel, Switzerland) and phosphatase inhibitor cocktail (Roche Diagnostics). Homogenized. For GLUT4 analysis, subcellular fractionation was performed and the plasma membrane (PM) fraction was collected (Mem-PER Plus membrane protein extraction kit, Thermo Fisher Scientific). Protein concentration was determined using the BCA protein assay kit (Pierce, Rockland, IL, USA).
Each lane of an 8% (for Stat3), 10% (for Akt and GLUT4), or 15% (for IL-6 and β-actin) Tris-glycine SDS-PAGE gel contains 30 µg of total protein. was included. Proteins were transferred to polyvinylidene difluoride membranes (Immobilen-P, Merck Millipore, Tokyo, Japan) and incubated with 3% nonfat dry milk in 10 mM Tris-HCl (pH 7.5)/150 mM NaCl containing 0.05% Tween 20. I blocked it. (TBST) 1 hour at room temperature. Membranes were incubated overnight at 4 °C in TBST containing 0.1% nonfat dry milk (w/v) and a 1:2000 dilution of the following primary antibodies: rabbit anti-Akt (Cell Signaling Technology, Danvers, MA, USA); ); rabbit anti-phospho Akt (Ser473) (Cell Signaling Technology); rabbit anti-Stat3 (Cell Signaling Technology); rabbit anti-phospho Stat3 (Thr705) (Cell Signaling Technology); rabbit anti-Glut4 (Cell Signaling Technology); IL-6 (Sigma-Aldrich); and mouse anti-β-actin (MBL Life Sciences, Tokyo, Japan). Horseradish peroxidase-conjugated goat anti-rabbit IgG (H + L) (1:20,000; Epitomics, Burlingame, CA, USA) was applied as the secondary antibody. Chemiluminescence was quantified using Western BLoT Quant HRP substrate (Takara Bio, Shiga, Japan) followed by image analysis (ImageQuant LAS-4000, GE Healthcare, Piscataway, NJ, USA). The amounts of phospho-Akt, phospho-Stat3, IL-6, and GLUT4 in the PM fraction were quantified using densitometry compared to total Akt, Stat3, β-actin, and GLUT4, respectively (Image J, National Institutes of Health), Bethesda, Maryland, USA).
IL-6 levels in plasma samples (prepared as described above) were quantified using a rat IL-6 ELISA kit (Sigma-Aldrich).
L6 rat myoblast cell line was obtained from Riken Cell Bank (Tsukuba, Japan), and cells were maintained in growth medium (Dulbecco’s modified Eagle medium). [DMEM]) containing 10% fetal bovine serum, GlutaMAX (Thermo Fisher Scientific), and 1% antibiotic antimycotic solution (Nacalai Tesque, Kyoto, Japan) at 37 °C under 5% CO2 Now on air. To maintain an undifferentiated state, L6 cells were passaged before reaching 80% confluence. Differentiation of L6 cells was induced by replacing the growth medium with differentiation medium (DMEM with 5% horse serum). [Thermo Fisher Scientific] and 1% antibiotic-antifungal solution) at 100% confluence. To ensure complete differentiation, cells were exposed to differentiation medium for 4 days before being used in experiments.
To obtain satellite cells, we isolated extensor digitorum longus muscles from male rats (8 weeks old) and used DMEM containing 0.2% type I collagenase (Wako Chemical), GlutaMAX, and 1% antibiotic/antimycotic solution. I digested it. The collected myofibers were confirmed to be free of contaminating cells such as fibroblasts under a high-power stereomicroscope. Satellite cells were obtained from isolated myofibers and cultured in mitogen-rich medium (DMEM containing 30% fetal bovine serum, GlutaMAX, and 1% chick embryo extract).[USBiological, Salem, MA, USA]10 ng/ml recombinant human basic fibroblast growth factor [154 amino acids, Nacalai Tesque, Kyoto, Japan], and 1% antibiotic-antimycotic solution) were applied to iMatrix-511 coated dishes (Matrixome, Osaka, Japan).Cells were confirmed to express Pax719,20. Cells were incubated at 37°C under 5% CO2.2 The medium was changed every two days in air.
L6 cells and satellite cells were preincubated for 18 h in growth medium containing 5 mM glucose (basal medium), followed by growth medium containing 10 mM glucose, 25 mM glucose, or 200 μM fatty acid (palmitic acid, Wako Chemical Industries). and incubated for 24 hours. . After this treatment, cells were collected in Sepasol-RNA I Super G (Nacalai Tesque) and stored at −80 °C until RNA extraction.
After 24 weeks, gastrocnemius and soleus muscle tissues were removed from CP or SP rats (n= 10 pieces per group). Total RNA was extracted using Sepasol-RNA I Super G solution (Nacalai Tesque). First strand cDNA was synthesized from total RNA using a commercially available kit (PrimeScript RT reagent kit; Takara Bio Inc.). The obtained samples were subjected to quantitative PCR analysis. Atrogine 1, Bnip3, casitas B lineage lymphoma proto-oncogene b (CBLB), Cathepsin L(CTSL), CD11c, CD68 , interleukin(illinois )-1β, IL-6 , LC3, Muscle-specific ring finger protein 1(MuRF1 ), Suppressor of cytokine signaling 3(SOCS3 ) and tumor necrosis factor(T.N.F. )-α. Quantitative PCR assays were performed on a StepOnePlus real-time PCR system (Applied Biosystems, Waltham, CA, USA) using SYBR Premix Ex Taq II reagents (Takara Bio). The relative abundances of all reaction products were normalized to the following levels: Hypoxanthine phosphoribosyltransferase I(HPRT ) mRNA calculated using the 2-delta delta CT method, independent of experimental factors.twenty one.
Additionally, total RNA was extracted from L6 and isolated satellite cells, transcribed into cDNA, and analyzed by quantitative PCR as described for skeletal muscle. The relative abundance of each reaction product was normalized to the following levels: β-glucuronidase(Gasbu) mRNA (for L6 cells) or Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activating protein(Yuwazu) mRNA (for satellite cells). Primer sets for all these genes are shown in Table 1.
I compared groups of data using Student t– One-way or two-way ANOVA with tests, Mann–Whitney U test, and post hoc Tukey–Kramer test (Prism 8, GraphPad Software). PValues less than 0.05 were considered significant. Data are reported as mean ± SEM.