Interactive Effects of Zinc and Zilpaterol Hydrochloride on Bovine β-Adrenergic Receptors

The objective of this study was to determine if the addition of zinc (Zn) in combination with zilpaterol HCL (ZH) affected the interaction of ZH with the beta2-adrenergic receptor (β-AR) by altering cAMP production, gene expression, and protein abundance in cultured skeletal muscle cells. Cultures of muscle bovine satellite cells were established and treated at 120 h with: 1) 0 μM Zn/zilpaterol hydrochloride (ZH; CON); 2) 0 μM Zn/10 μM ZH (ZH); 3) 1 μM Zn from Zn chloride/0 μM ZH (Zn); 4) 1 μM Zn from Zn chloride/10 μM ZH (ZN/ZH) in differentiation media for an additional 0, 6, 24, 48 and 96 h. Protein and mRNA were isolated and quantified at 24 and 96 h, and cAMP was measured at 0, 6, 24, 48 and 96 h. At 0, 24, 48 and 96 h, no differences (P > 0.05) were detected in cAMP production. At 6 h, Zn cells had the greatest concentration of cAMP (P < 0.05) compared to ZH treatments. No differences (P > 0.05) were detected in mRNA abundance at 24 h. At 96 h, 0 μM Zn/10 μM ZH cells had an increased abundance of myosin heavy chain (MHC)-I mRNA (P < 0.05) compared to CON. Furthermore, ZH had a greater abundance of MHC-IIX mRNA (P < 0.05) and a tendency for a greater abundance of IGF-1 mRNA (P < 0.15) compared to CON and ZN/ZH. No differences (P > 0.05) were detected in the protein abundance of β1AR and the β2AR. These results indicated Zn and ZH in combination did not have an additive effect on β2-AR function as indicated by cAMP concentrations.

industry to improve growth performance and carcass characteristics through increased protein synthesis and decreased protein degradation [1]. Beta-adrenergic agonists have also been reported to increase lipolysis and decrease lipogenesis in adipose tissue [1] [2] [3]. These β-AA work through an interaction with the beta-adrenergic receptors (β-AR) [4] [5]. Zilpaterol HCl (ZH), a β-AA used in cattle, primarily binds with the β 2 -AR, which is the most predominant β-AR found in cattle muscle and adipose tissue [1] [2]. Via a secondary messenger signal cascade event, cyclic adenosine monophosphate (cAMP) is activated thereby resulting in protein accretion and lipid catabolism [1] [2] [3].
Overstimulation of the β-ARs by β-AA has been reported to result in receptor desensitization [6] [7]. Receptor desensitization results in a down regulation of adenylate cyclase catalytic activity resulting in a reduction of cAMP synthesis [8]. When the β-ARs become desensitized, they are sequestered within an intracellular vesicle, thus losing the ability to induce signal transduction [6] [7].
Research has shown that the β 2 -AR potentially have multiple allosteric binding sites for zinc (Zn) [1] [9]. Swaminath, Lee and Kobilka [10], suggested there are two main binding sites for Zn on the β-AR; one affects the agonist's ability to bind to the receptor, while the other affects the antagonist's ability to bind to the receptor thus increasing cAMP production. Zinc also regulates adenylate cyclase (AC) and cyclic nucleotide phosphodiesterase (PDE) which are involved in the synthesis and degradation of cAMP after the β-AR is activated [11]. Several studies have reported that the catalytic activity of AC is inhibited by Zn; however, the mechanism responsible for this phenomenon is still unknown [12] [13] [14]. von Bülow, Rink and Haase [15] reported the addition of Zn to cellular lysate inhibits cyclic nucleotide degradation, signifying increases in cellular Zn will block PDE activity.
Little is known about how the combination of ZH and Zn might influence the β-AR's ability to produce cAMP, and its regulation of mRNA and protein synthesis. Thus, the objective of the present research was to determine if utilizing Zn in combination with ZH would affect the downstream signal transduction of cascade events commonly associated with β-AA thus altering cAMP activation, and mRNA and protein abundance.

Experimental Design and Treatments
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.

Satellite Cell Isolation
Satellite cell isolation was performed following procedures outlined by Johnson et al. [6].

Satellite Cell Culture
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 matri-  Table 1. Assays were performed in the Ge-neAmp 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).

Protein Extraction and Western Blots
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 Na 3 VO 4 (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 Table 1. Sequence of bovine-specific PCR primers and TaqMan probes to be used for determination of expression of mRNA of AMPKα, MHC-I, MHC-IIA, MHC-IIX, IGF-I,   β1AR, β2AR, β3AR, CEBPβ, GPR43, GPR41, Glut4, PPARγ, SCD and RPS9*.

cAMP Isolation and ELISA
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.

Statistical Analysis
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.

Results and Discussion
At 0, 6, 24, 48, and 96 h of incubation, cAMP was measured with no difference observed between treatments at 0, 24, 48, and 96 h (P > 0.05; Table 2). However,  Table 2). This is in contrast to that reported by [16], who reported no difference in cAMP concentration at 6 h between bovine satellite cells treated with Zn and ractopamine HCl (RH). In the Harris [16] study, using Zn and RH, cells treated with 1 µM Zn/10 µM RH exhibited the greatest cAMP concentration at 24 h and by 96 h the control group had a greater concentration of cAMP compared to the cells treated with RH only [16]. Ractopamine HCl is β-AA used in beef and pork production that primarily binds to β1AR [17]. Ractopamine HCl does not affect bovine cells to the extent as ZH because the majority of the β-AR are β2AR [1]. Klein, Sunahara, Hudson, Heyduk and Howlett [13] reported decreased concentrations of cAMP in N18TG2 Neurblastoma cells treated with 300 µM Zn 2+ and forskolin or PGE 1 for 2 h. In the study, cells treated only with Zn 2+ , resulted in no effect on cAMP concentration [13]. Swaminath, Steenhuis, Kobilka and Lee [9] reported Zn binds to the β2AR, causing increased agonist affinity and a greater production of cAMP. Swaminath, Lee and Kobilka [10] further reported multiple binding sites on the β2AR for Zn, with the most prominent binding site for Zn causing an increase in agonist binding affinity and a decrease in antagonist affinity.
Relative mRNA abundance of β1AR, β2AR, AMPKα, IGF-1, MHC-I, MHC-IIA, MHC-IIX, GPR43, SCD, CEBPβ, and PPARγ yielded no difference between treatments at 24 h (P > 0.05; Table 3). At 96 h, ZH cells tended to increase the abundance of MHC-I mRNA (P < 0.10; Table 3) compared to CON. Furthermore, ZH cells had a greater abundance of MHC-IIX mRNA (P < 0.05; Table 3) and a tendency for greater abundance of IGF-I mRNA (P < 0.15; Table 3) compared to CON and Zn/ZH. Harris [16] reported no differences in β1AR, β2AR, and Johnson [2] however, reported a decrease in β1AR and β2AR mRNA abundance compared to control bovine cells, when cells were treated with 1 µM ZH. Tokach [18] found bovine cells treated with ZH increased the abundance of IGF-I mRNA; however, ZH decreased MHC-I mRNA abundance and increased MHC-IIX mRNA abundance compared to control cells at 120 h. In the current study, ZH increased the abundance of MHC-IIX mRNA (P < 0.05) and tended (P < 0.15) to increase the abundance of MHC-I mRNA compared to control cells at 96 h.
Protein abundance of β1AR and β2AR showed no difference between treatments at either 24 or 96 h (P > 0.05; Table 4). Our data support that of [16], who  Table 3. Relative alterations of mRNA concentrations of AMPKα, IGF-I, MHC-I, MHC-IIA, MHC-IIX, β1AR, β2AR, GPR43, SCD, CEBPβ, and PPARγ genes in bovine skeletal muscle satellite cells treated with zinc (Zn) and zilpaterol hydrochloride (ZH).  consequently result in an inhibition of the synthesis of cAMP. The cAMP data indicated that 1µM Zn/10 µM ZH may be inhibiting the production or accelerating the degradation of cAMP. Lynch, Patson, Goodman, Trapolsi and Kimball [19] reported that Zn became inhibitory to cell growth at concentrations over 100 µM. When β-AAs bind to a β-AR, intrinsic Zn is released. With ZH having a high affinity to bind to β2ARs, which is the predominant β-AR found in beef cattle muscle and adipose tissue, and the β2AR potentially having multiple allosteric binding sites for Zn [9], the cell may be flooded with Zn from intrinsic and free sources of Zn. This may in part cause Zn to become inhibitory towards AC thus reducing the amount of cAMP produced. Since cAMP is a secondary messenger in the β-AR pathway that leads to an increase in myogenic mRNA transcription and ultimately muscle protein accretion, this could possibly explain the decreased myogenic activity we observed. However, large concentrations of Zn increase the uptake of glucose and de novo lipogenesis [19], possibly partially elucidating the reason for increased adipogenic activity observed in this study.
Based on the results of this study, we can conclude that independently, Zn and ZH positively impact myogenic synthesis; however, cAMP production, β-AR protein and mRNA abundance may not be affected by the combination of the two compounds. Increasing Zn supplementation may increase the concentration of extracellular free Zn; possibly increasing the binding affinity of the β-AA, therefore amplifying the signal transduction associated with β-AA. This amplified affect may result in over stimulation of the β-AR, thereby activating AC causing an increased release of intracellular Zn, which could negatively impact cAMP. While there is conflicting evidence on the implications between the interactions of Zn, β-AA and β-AR, these mechanisms are not fully understood, and future research should be conducted to further elucidate the molecular mechanisms that impact cellular muscle metabolism in biological processes involving Zn. Caution should be used extrapolating these in vitro results to expected results of feeding ZH to beef cattle.

Supported
Supported in part by funding from Zinpro Corporation, Eden Prairie, Minneso-