This experiment was conducted as a 2 × 2 factorial, and each replicate (n = 4) was plated and cultured simultaneously. These experiments were conducted in 2017 in the Department of Animal and Food Sciences at Texas Tech University. The cells were treated with a laboratory grade Zn chloride (Acros Organics, Fisher Scientific, Fair Lawn, NJ), ZH, or a combination of the two. Each well was randomly assigned to one of four treatments: 1) 0 µM Zn/0 µM ZH (CON); 2) 0 µM Zn/10 µM ZH (ZH); 3) 1 µM Zn/0 µM ZH (Zn); 4) 1 µM Zn/10 µM ZH (ZN/ZH).

Satellite cell isolation was performed following procedures outlined by Johnson et al. [6]. Muscle tissue samples were extracted from the semimembranosus muscle of market age cattle at harvest. Tissue was then subjected to satellite cell extraction procedures, to isolate satellite cells from muscle tissue. Under a sterile hood, adipose and connective tissue were removed from muscle. The muscle was ground through a sterile grinder sterilized in 70% ethanol for 24 h prior to use. Ground muscle was then incubated in a solution consisting of 0.1% pronase (Calbiochem, La Jolla, CA) and Earl’s Balanced Salt Solution (EBSS; Sigma, St. Louis, MO) for 1 h at 37˚C. At 10 min intervals, the samples were shaken vigorously. Following incubation, differential centrifugation at 1500 × g for 4 min at 25˚C was performed on the sample. The resulting supernatant was removed, and the pellet suspended in phosphate buffered saline (PBS; Invitrogen, Grand Island, NY; 140 mM NaCl, 3 mM Na₂-H-PO₄). The resulting pellet was then centrifuged at 500 × g for 10 min at 25˚C. The supernatant was transferred into another container and centrifuged at 15,000 × g for 10 min at 25˚C to form a pellet consisting of mononucleated cells. The differential centrifugation and the PBS wash steps were repeated twice. Total mononucleated cells were then suspended in cold Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen, Grand Island, NY) containing 10% fetal bovine serum (FBS; Invitrogen) and 10% dimethylsulfoxide (DMSO; Sigma). The cell solution was then aliquoted into 1.8 mL nunc cryo tube vials (Fisher Scientific). Cells were then placed in a −80˚C freezer for 24 h and stored in liquid nitrogen until needed.

Bovine satellite cells were cultured in 6-well plates (RNA and Protein analysis) or 24-well plates (cAMP analysis). Plates were coated with reduced factor matrigel (Matrigel; BD Biosciences, Bedford, MA) at least 1 h prior to plating cells and kept at 37˚C. Cells were plated and placed in a 37˚C incubator for 24 h in 10% Fetal Bovine Serum (FBS; GIBCO^Ò; Invitrogen)/DMEM-3X antibiotic antimycotic (Invitrogen) 0.3X gentamycin (Sigma) media. Cells were rinsed and allowed to proliferate for 120 h in 10% FBS/DMEM-3X antibiotic antimycotic 0.3X gentamycin at 37˚C. At 120 h media was changed from proliferation to differentiation media (3% Horse Serum; GIBCO^Ò; Invitrogen)/DMEM-3X antibiotic antimycotic 0.3X gentamycin). The treatment substrates were added to the differentiation media. Cells that were designated for mRNA and protein quantification were treated and incubated for either 24 or 96 h in differentiation media, while cells destined for cAMP analysis were treated and incubated for a total of 0, 6, 24, 48, or 96 h in differentiation media.

At 24 or 96 h of treatment, cells from 6-well plates were harvested for mRNA analysis. The cells were rinsed 3 times in PBS, and total mRNA isolated with ice-cold buffer containing TRI Reagent^Ò (Sigma, St. Louis, MO). Approximately 200 mL of TRI Reagent^Ò were added to each well, and then incubated for 5 min at 25˚C. The wells were then scraped to ensure release of all cells from the bottom of the well. Homogenate was pipetted into a microcentrifuge tube, 100 µL of chloroform were added, vortexed for 30 s and incubated for 5 min at 25˚C. The sample was then centrifuged at 15,000 × g for 15 min that causing the samples to separate into 3 layers. The top supernatant layer was pipetted off and placed into a new microcentrifuge tube. Ice cold isopropyl alcohol (250 μL) was added to the supernatant, shaken, and incubated for 10 min at 25˚C. Samples were centrifuged at 15,000 × g for 10 min. The supernatant was poured off, the RNA pellet at the bottom of each tube air dried, and 500 µL of 75% ethanol was added to each tube to rinse and suspend the RNA pellet. Samples were then placed in a −80˚C freezer until needed (no longer than 3 months). Samples were removed from the freezer and thawed on ice and centrifuged at 250 × g for 10 min, ethanol poured off, and the pellet air dried. Nuclease free water (30 μL) was added to dissolve the RNA pellet. The concentration of RNA was determined with a spectrophotometer at an absorbance of 260 nm using a NanoDrop 1000 (NanoDrop products, Wilmington, DE). Samples were treated with DNAse to remove any DNA contaminants using a DNA-free kit (Life Technologies). The RNA was then subjected to reverse-transcription to produce cDNA. The resulting cDNA was used for real-time quantitative reverse transcription-PCR (RT-qPCR) to measure the abundance of AMP-activated protein kinase alpha (AMPKα), beta-1 adrenergic receptor (β1AR), beta-2 adrenergic receptor (β2AR), insulin-like growth factor-I (IGF-I), myosin heavy chain (MHC)-I, MHC-IIA, MHC-IIX, C-enhancer binding protein beta (CEBPβ), G-protein coupled receptor 43 (GPR43), peroxisome proliferator-activated receptor gamma (PPARγ), and stearoyl-CoA desaturase (SCD) mRNA relative to the abundance of ribosomal protein subunit 9 (RPS9) mRNA in total RNA isolated from cells. Bovine primers and probes for AMPKα, β1AR, β2AR, IGF-I, MHC-I, MHC-IIA, MHC-IIX, GPR43, SCD, CEBPβ, and PPARγ are presented in Table 1. Assays were performed in the GeneAmp 7900HT Sequence Detection System (Applied Biosystems, Life Technologies) using thermal cycling parameters recommended by the manufacturer (40 cycles of 15 s at 95˚C and 1 min at 60˚C).

At 24 or 96 h of treatment, cells from 6-well plates were harvested for protein analysis. The cells were rinsed 3 times with PBS. Protein from cells was isolated with ice-cold buffer containing mammalian protein extraction reagent (M-PER; Fisher Scientific, Fair Lawn, NJ), protein inhibitor (Roche, Branchburg, NJ), and 2 mM Na₃VO₄ (Fisher Scientific). Approximately 500 µL of M-PER was added to each well and incubated for 5 min at 25˚C while shaking. The wells were then scraped to ensure all cells were released from the bottom of the well. Samples

*AMPKα = AMP-activated protein kinase alpha, MHC-I = myosin heavy chain-I, MHC-IIA = myosin heavy chain-IIA, MHC-IIX = myosin heavy chain-IIX, β1AR = beta 1 adrenergic receptor, β2AR = beta 2 adrenergic receptor, β3AR = beta 3 adrenergic receptor, CEBPβ = C-enhancer binding protein beta, GPR43 = G-protein coupled receptor 43, GPR41 = G-protein coupled receptor 41, Glut4 = glucose transporter type 4, PPARγ = peroxisome proliferator-activated receptor gamma, SCD = stearoyl-CoA desaturase and RPS9 = ribosomal protein S9.

were centrifuged at 1500 × g for 15 min., separating the sample into 3 layers. The middle supernatant layer was removed and placed into a microcentrifuge tube. Protein samples were then diluted with either M-PER to determine protein concentration using the Pierce^TM BCA^TM protein assay (Thermo Fisher Scientific, Fairlawn, NJ). Protein concentration was then determined using a NanoDrop 1000 spectrophotometer (Thermo Scientific, Wilmington, DE) at 562 nm. All samples were then diluted to the same concentration. Modified Wangs tracking dye was added to samples for western blot analysis. Samples were denatured with β-mercaptoethanol and incubated for 2 min at 95˚C. Samples were then loaded onto Novex 4% - 12% Bis-Tris gels (Invitrogen), and protein was separated by gel electrophoresis run for approximately 35 min at 165V and 27 mA. Proteins were transferred onto a nitrocellulose membrane (Invitrogen) for 7 min. Following transfer, the membrane were incubated with non-fat dry milk (BIO RAD, Hercules, CA), 10% 10 × Tris-buffered saline (TBS) in NanoPure water for 1 h at 25˚C to block non-specific antibody binding. The blocking solution was then removed from the membrane. The appropriate primary antibody: 1:1000 α-beta 1 AR, rabbit, IgG (abcam^Ò, Cambridge, MA); 1:1000 α-beta 2 AR, goat, IgG (abcam) was mixed into 1 × TBS-Tween solution, added to the membrane and allowed to incubate for 2 h (β1AR) or 1 h (β2AR) at 25˚C. The membrane was then rinsed 3 times for 10 min in TBS-Tween. The appropriate Alexa fluorescent antibodies: goat α-rabbit, IgG, Alexa-Fluor 633 (Invitrogen); donkey α-goat, IgG, Alexa-Fluor 633 (Invitrogen) were then added at a dilution of 1:2000 in TBS-Tween to the membrane and incubated for 1 h at 25˚C in the absence of light. The membranes were then rinsed 3 times for 10 min in TBS-Tween in unlighted conditions. The membranes were then dried and visualized using Imager Scanner II and ImageQuant TL software. Densitometry measurements were made on the bands corresponding to β1AR and β2AR using a molecular weight standard for reference (Precision Plus Protein^TM All Blue Standards; BIO RAD).

After 0, 6, 24, 48, and 96 h, cells from 24-well plates were harvested for cAMP analysis. Cells were rinsed 3 times in PBS. Then 100 µL of 0.1 M HCl was used to lyse the cells. Cells were incubated for 5 min at 25˚C while shaking. The wells were then scraped to ensure all cells were lysed and released from the bottom of the well. The sample was taken from the wells and placed into microcentrifuge tubes. An enzyme-linked immunosorbent assay (ELISA; Sigma, St. Louis, MO) was performed on samples to determine cAMP concentration, following instructions provided by the manufacturer. The results were read with a Spectra max 380pc plate reader and Softmax Pro software.

Data were analyzed using the GLIMMIX procedure of SAS (v.9.3, SAS Institute; Carey, NC). The model included treatment as the fixed effect, and the Kenward-Roger adjustment was used to correct degrees of freedom. Means were separated using the LSMEANS procedure PDIFF option and considered different when P ≤ 0.05. Tendencies for differences among treatment means were declared when 0.05 < P ≤ 0.15.