2.1 Animals and Treatments. The details of the study design were adapted from previously published work (19). Briefly, 28 Sprague-Dawley rats were assigned to four groups: regular diet (CON; n=7), high-fat diet (HFD; n=7), geranylgeraniol+high-fat diet (GG; n=7), and ginger root extract+high-fat diet (GRE; n=7). CON was given an AIN-93G diet (10% calories from fat) throughout the eight weeks of the study. HFD were fed a high-fat diet (45% calories came from fat, Research Diets, Catalog # 12451), while GG was given a high-fat diet with 800 mg/kg GGOH (American River Nutrition, LLC., Hadley, MA), and GRE was given a high-fat diet with 0.75% ginger (Ginger root extract 20%, Lot #G190297, Sabinsa Corporation, East Windsor, NJ) for eight weeks. After two weeks of feeding, HFD, GG, and GRE were given a streptozotocin (STZ) dose of 35 mg/kg to induce diabetes. Fasting blood sugar was collected, and rats were considered diabetic if fasting blood sugar was above 200 mg/dl. The HFD, GG, and GRE rats were confirmed to have diabetes based on their fasting glucose levels (data not shown). All rats were kept in individual cages with the temperature set at 21 ± 2°C with a 12h light-dark cycle. They were fed their respective diets twice a week and had free access to food and water. The Texas Tech University Health Sciences Center Institutional Animal Care and Use Committee approved all conditions and handling of the animals. All experiments were performed by the relevant guidelines and regulations.
2.2 Sample Collection. Blood and soleus muscle were collected from the rats after being fasted for 4 hours and stored at −80°C for further analysis. In addition, the right and left soleus muscles were harvested to perform gene and protein expression and immunohistochemical analysis.
2.3 RNA Isolation and RT-qPCR Analysis. A detailed description of muscle tissue homogenization and RNA isolation has been published previously (37). RNA was isolated from flash-frozen muscle samples (~30 mg) using the RNeasy Fibrous Tissue Mini Kit (Cat. # 74704, Qiagen, MD) with the recommended standardized protocol. cDNA was synthesized from 1 ug of total RNA using an Iscript reverse transcriptase kit (Bio-Rad). Real-time PCR amplification experiments and calculations of relative expression levels were performed following the user manual # 2 ABI PRISM70500 Sequence Detection System (Applied Biosystems) with iTag SYBR Green Supermix (Bio-Rad, CA). Pre-designed assays for Pax7 and MyoD were obtained from Integrated DNA Technologies with beta-actin (Act-b) as the internal control. Duplicate technical replications were performed for each assay. Data were then analyzed using the 2-ΔΔCt method to determine relative fold change in transcript abundance.
2.4 Muscle Gene Expression. Muscle samples were analyzed for intramuscular gene expression of Pax7 and MyoD for CON, HFD, GG, and GRE. Real-time PCR was performed using # 2 ABI PRISM70500 Sequence Detection System (Applied Biosystems) with iTag SYBR Green Supermix (Bio-Rad, CA)and was normalized to β-actin. The 2-ΔΔCt method was used to determine relative fold change in transcript abundance.
2.5 Immunohistochemistry. Muscle cross-sections (7 μm) were stained with antibodies against, Dystrophin (1:100; Pa5-32388; Thermofisher, MA), MHC1 (1:500; NBP2-50298; Novus, CO), MyoD (1:100; 18943-1-AP; Proteintech, IL), Myostatin (MSTN; 1:1000; AB3239-1 Millipore, MA), and Pax7 (1:100; SC-81648; Santa Cruz, TX). Appropriate secondary antibodies were applied: anti-rabbit (1:1000; ab50081; Thermofisher, MA) and anti-mouse (1:1000; R6393; Thermofisher, MA). Histochemical methods were adapted from previously published methods (25,26). Briefly, for co-immunofluorescence staining (Dystrophin and Pax7; Dystrophin, MyoD, and Pax7; Dystrophin, MSTN, and Pax7), sections were fixed with ice-cold acetone for 5 min followed by washing in PBS. Next, sections were blocked with 2% H2O2 for 10 min and then 1% BSA solution for 20 min. Following blocking, sections were incubated in the primary antibody at 4ºC overnight. After washing, sections were then incubated in the appropriate secondary antibodies. Sections were then re-fixed in ice-cold acetone to prevent migration of the secondary antibodies and re-blocked. The sections were then incubated in the second primary antibody, followed by incubation in the appropriate secondary antibody. Sections were then washed with PBS and 4′,6-diamidino-2-phenylindole (DAPI; D1306; Invitrogen, MA) for nuclear staining. Slides were then visualized using a Zeiss Axiovert 200m Inverted Fluorescent Motorized Microscope. Images were taken, and the number of cells expressing SCs was counted and normalized to 100 fibers. Additionally, muscle CSA was analyzed using Image J (National Institute for Health, Bethesda, MD, USA) for all samples. One hundred muscle fiber areas were measured for each sample.
2.6 Tissue Homogenization and Western Blot Analysis. The details of the western blot procedure were adapted from previously published work (19). Briefly, frozen muscle samples were homogenized in 10 mL/mg muscle of ice-cold RIPA buffer (89901; Sigma Aldrich, MO) containing 1X protease inhibitor (1:100; PPC1010; Sigma Aldrich, MO). Homogenate was collected, analyzed for total protein concentration using Pierce™ BCA Protein Assay Kit (23225; Thermofisher, MA). Aliquots from homogenates were loaded (equal amount of protein) per lane in duplicate and separated by SDS-PAGE. All proteins were run on 10% gels (4561033; Bio-Rad, CA) for 60 min at 120 V. Samples were then transferred to a Polyvinylidene fluoride membrane for immunoblotting via electrophoresis for 150 min at 70 V. The ladder on the membranes was then marked with WesternBright ChemiPen (R-07055-001; Advansta Inc.,CA). Next, Non-Fat Milk Powder was used to block the membranes, followed by primary antibodies incubation with Pax7 (1:1000; sc-81648; Santa Cruz, TX), MyoD (1:2000; 18943-1-AP; ProteinTech, IL), MSTN (1:1000; AB 3239-1; Millipore Sigma, MA), and GAPDH (1:4000; 8884; Cell Signaling, MA) overnight. Then, membranes were incubated with secondary anti-mouse IgG (1:1000; 7076S Cell Signaling, MA) and anti-rabbit IgG (1:1000; 7074S; Cell Signaling, MA) for 60 min accordingly. Chemiluminescent substrate (K-12043-D10; Advanta Inc., CA) and the ChemiDoc MP Imaging System (Bio-Rad, CA) was used to picture the stained protein bands. Next, membranes were stripped using 5X Western Reprobe for 90 min. Image Lab Software (12003154; Bio-Rad, CA) was used to analyze band density. Total protein concentrations were normalized to a housekeeping protein GAPDH and expressed as arbitrary units.
2.7 Statistical Analyses. Statistical analyses were performed using SPSS (IBM version 29; Armonk, NY: IBM Corp) for all analyses. Two separate one-way ANOVAs were used to analyze gene expression, protein content, soleus muscle CSA and satellite cell numbers for the supplemental groups. In addition, Bonferroni post hoc tests were used for pairwise comparisons. The statistical significance was accepted at p < 0.05. Data are reported as mean ± SE.