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E. K. Miller, K. Y. Chung, J. P. Hutcheson, D. A. Yates, S. B. Smith and B. J. Johnsonadipose tissue explants

cells but has little effect on de novo fatty acid biosynthesis in bovine subcutaneous-adrenergic receptors in bovine muscleZilpaterol hydrochloride alters abundance of

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Zilpaterol hydrochloride alters abundance of -adrenergic receptorsin bovine muscle cells but has little effect on de novo fatty acid

biosynthesis in bovine subcutaneous adipose tissue explants

E. K. Miller,* K. Y. Chung, J. P. Hutcheson, D. A. Yates,S. B. Smith, and B. J. Johnson1

*Department of Animal Sciences and Industry, Kansas State University, Manhattan 66506;Department of Animal and Food Sciences, Texas Tech University, Lubbock 79409;

Intervet Schering Plough Animal Health, De Soto, KS 66018;and Department of Animal Science, Texas A&M University, College Station 77843

ABSTRACT: We predicted that zilpaterol hydrochlo-ride (ZH), a -adrenergic receptor (AR) agonist, woulddepress mRNA and protein abundance of -AR in bo-vine satellite cells. We also predicted that ZH would de-crease total lipid synthesis in bovine adipose tissue. Bo-vine satellite cells isolated from the semimembranosusmuscle were plated on tissue culture plates coated withreduced growth factor matrigel or collagen. Real-timequantitative PCR was used to measure specific geneexpression after 48 h of ZH exposure in proliferatingsatellite cells and fused myoblasts. There was no effectof ZH dose on [3H]thymidine incorporation into DNAin proliferating myoblasts. Zilpaterol hydrochloride at1 Mdecreased (P< 0.05) 1-AR mRNA, and 0.01and 1 MZH decreased (P< 0.05) 2-AR and 3-ARmRNA in myoblasts. The expression of IGF-I mRNAtended to increase (P= 0.07) with 1 MZH. There wasno effect (P> 0.10) of ZH on the -AR or IGF-I gene

expression in fused myotube cultures at 192 h or on

fusion percentage. The 2-AR antagonist ICI-118, 551at 0.1 Mattenuated (P< 0.05) the effect of 0.1 MZH to reduce expression of 1- and 2-AR mRNA. Thecombination of 0.01 MZH and 0.1 MICI-118, 551caused an increase (P< 0.05) in 1-AR gene expres-sion. There was no effect (P> 0.10) of ICI-118, 551 orZH on 3-AR or IGF-I. Western blot analysis revealedthat the protein content of 2-AR in ZH-treated myo-tube cultures decreased (P< 0.05) relative to control.Total lipid synthesis from acetate was increased by ZHin bovine subcutaneous adipose tissue explants in theabsence of theophylline but was decreased by ZH whentheophylline was included in the incubation medium.These data indicate that ZH alters mRNA and proteinconcentrations of -AR in satellite cell cultures, whichin turn could affect responsiveness of cells to prolongedZH exposure in vivo. Similar to other -adrenergic ago-nists, ZH had only modest effects on lipid metabolism

in adipose tissue explants.Key words: adipose tissue, beta-adrenergic receptor, bovine, satellite cell, zilpaterol hydrochloride

2012 American Society of Animal Science. All rights reserved. J. Anim. Sci. 2012. 90:13171327http://dx.doi.org/10.2527/jas.2011-4589

INTRODUCTION

Zilpaterol hydrochloride (ZH) is an orally active-adrenergic receptor (AR) agonist approved for usein feedlot cattle in the United States (Delmore et al.,2010). Administration of ZH to feedlot steers and heif-

ers the last 20 to 40 d on feed increased ADG, ribeyearea, and dressing percentage and improved feed ef-ficiency and carcass yield grade (Beckett et al., 2009;

Montgomery et al., 2009; Baxa et al., 2010). Zilpaterolhydrochloride elicits a response through binding to-AR, which are membrane-bound receptors locatedon most mammalian cells (Strosberg et al., 1993; Millsand Mersmann, 1995). The activation of protein kinaseA, as elicited by -adrenergic agonist (-AA), is re-sponsible for changes in protein synthesis and degrada-tion, particularly in skeletal muscle (Mersmann, 1998),and ZH putatively works to increase muscle growth viabinding to the -AR. The treatment of ZH induced fastglycolytic fiber types such as myosin heavy chain IIXrather than slow oxidative fiber types such as myosinheavy chain I (Baxa et al., 2010).

There is limited information on the direct effectsof ZH on bovine skeletal muscle and adipose tissue.

1Corresponding author: [email protected] August 12, 2011.Accepted November 4, 2011.

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Therefore, one purpose of these experiments was toinvestigate the direct effects of ZH on bovine satel-lite cell proliferation and the expression of mRNA forthe -AR subtypes and IGF-I in bovine satellite cellcultures. Marked reductions in subcutaneous (s.c.) fatthickness were noted in sheep fed cimaterol or clen-buterol (Baker et al., 1984; Thornton et al., 1985;Hamby et al., 1986). However, clenbuterol treatment

elicited a small increase in adipocyte size in lambs(Coleman et al., 1988). Ricks et al. (1984) first dem-onstrated a significant reduction in carcass fat from 24finishing steers. Subsequently, Miller et al. (1988) re-ported a 40% reduction in s.c. fat thickness and KPHin heifers fed clenbuterol. However, we reported nochange in amount of s.c. fat or s.c. fat thickness inyoung, clenbuterol-fed Angus steers (Schiavetta et al.,1990). Therefore, this study also documented short-term effects of ZH on lipid synthesis in bovine adiposetissue explants to establish whether ZH depresses adi-pogenesis via direct effects on adipose tissue metabo-lism.

MATERIALS AND METHODS

All experimental procedures with animals were ap-proved by the Kansas State University and the TexasA&M University Institutional Animal Care and Usecommittees.

Bovine Satellite Cell Isolation

Satellite cell isolation was conducted as describedpreviously (Johnson et al., 1998). Cattle were eutha-

nized by captive bolt stunning followed by exsangui-nation following standard industry procedures. Usingsterile techniques, approximately 500 g was dissectedfrom the semimembranosus muscle and transportedto the cell culture laboratory at Kansas State Uni-versity. Subsequent procedures were conducted in asterile field under a tissue culture hood. After removalof connective tissue the muscle was passed througha sterile meat grinder. The ground muscle was incu-bated with 0.1% pronase (CalBiochem, La Jolla, CA)in Earls balanced salt solution for 1 h at 37C withfrequent mixing. After incubation, the mixture wascentrifuged at 1,500 gfor 4 min at room tempera-

ture, the pellet was suspended in PBS (140 mMNaCl,1 mMKH2PO4, 3 mMKCl, 8 mMNa2HPO4), and thesuspension was centrifuged at 500 gfor 10 min. Thesupernatant fraction was centrifuged at 1,500 gfor10 min to pellet the mononucleated cells. The PBSwash and differential centrifugation were repeated 2more times. The resulting mononucleated cell prepa-ration was suspended in 4C Dulbeccos modified Ea-gle medium (DMEM) containing 10% fetal bovineserum (FBS) and 10% (vol/vol) dimethylsulfoxideand frozen. Cells from 7 animals were stored frozen inliquid nitrogen.

[3H]Thymidine Incorporation

Satellite cells were plated on 2-cm2 culture plates forthe measurement of thymidine incorporation into DNA.Culture plates were precoated with reduced growth fac-tor basement membrane matrigel diluted 1:10 (vol/vol)with DMEM. Cells were plated in DMEM containing10% FBS and incubated at 37C, 95% CO2 in a water-saturated environment. Plating density for cells wasempirically established so that all cultures were 25 to50% confluent after the incubation period. This ensuredthat cell proliferation rate was not affected by contactinhibition. Cultures were rinsed 3 times with serum-free DMEM 24 h after plating the bovine satellite cellsin 10% FBS/DMEM, and different concentrations ofZH (0, 0.0001, 0.001, 0.01, 0.1, 1.0, and 10 M; MerckAnimal Health, Whitehouse Station, NJ) were addedin 10% FBS/DMEM. At 72 h, cultures were rinsed 3times with serum-free DMEM and 1 Ci/mL of [3H]thymidine (NEN Life Science, Boston, MA) was addedto each well. Cells with [3H]thymidine were incubated

at 37C, 5% CO2 in a water-saturated environment for3 h. After incubation, satellite cells were rinsed 3 timeswith cold serum-free DMEM to remove free [3H]thymi-dine. Cold 5% trichloroacetic acid (Sigma, St. Louis,MO) was added to every well and incubated overnightat 4C. The next day, cells were rinsed 2 times with coldtrichloroacetic acid to remove any remaining unincor-porated [3H]thymidine. The precipitated cell materialwas dissolved in 0.5 mL of 0.5 M sodium hydroxide(NaOH; Sigma) in a rocking incubator for 30 min at37C. The NaOH suspensions were transferred quan-titatively into scintillation vials containing 10 mL ofscintillation cocktail (Fisher Scientific, Hanover Park,

IL). The samples were allowed to stand for a few hoursin low light to reduce chemiluminescence before beingcounted in a scintillation counter. All treatments weremeasured in triplicate, with 7 total assays included inthe final analysis.

Satellite Cell Differentiation

Satellite cells were plated as described previously on9.62-cm2 collagen-coated culture plates for differentia-tion studies. At 48 h, cultures were rinsed 3 times withserum-free DMEM and 3% swine serum/DMEM was

added. At 96 h, cells were rinsed 3 times with serum-free DMEM and 3% horse serum (HS)/BSA-linoleicacid (1.5 g/mL)/DMEM fusion media was added. Zil-paterol hydrochloride (0, 0.0001, 0.001, 0.01, 0.1, and 1M) was added to the cultures at 144 h. After approxi-mately 216 h in culture, cells fused into multinucleatedmyotubes and were stained using Hoechst 33342 stain.

RNA Isolation on Proliferating Myoblasts

Satellite cells were plated in 10% FBS/DMEM asdescribed previously in 9.62-cm2 tissue culture plates.

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After 48 h, cultures were 25 to 50% confluent andwere rinsed 3 times with serum-free DMEM and ZH(0, 0.0001, 0.001, 0.01, and 1 M) was added to thecultures in 10% FBS/DMEM. At 96 h, total RNAwas isolated using the Absolutely RNA Microprep Kit(Stratagene, La Jolla, CA). The concentration of RNAwas determined by absorbance at 260 nm. One micro-gram of total RNA was reverse transcribed to produce

first-strand cDNA using TaqMan Reverse Transcrip-tion Reagents, MultiScribe Reverse Transcriptase (Ap-plied Biosystems, Foster City, CA). Random hexamerswere used as primers in cDNA synthesis.

In addition, satellite cells were plated as describedpreviously to assess the effects of ZH alone or in com-bination with a specific 2-AR antagonist, ICI-118, 551(Sigma). Treatments consisted of the following: 1) con-trol, 2) 0.01 MZH, 3) 0.1 MICI, 4) 0.01 MZH +0.1 MICI, and 5) 0.01 MZH + 0.01 MICI. Theconcentration of inhibitor chosen to use in combinationwith ZH was similar to that in previous experiments weconducted in our laboratory with comparable experi-

ments in which receptor antagonists were used to in-hibit the effect of its agonist (Sissom et al., 2006). Cul-ture conditions and RNA isolation were as describedpreviously.

RNA Isolation from Myotubes

Bovine satellite cells were plated in 10% FBS/DMEM as described previously on 9.62-cm2 tissue cul-ture plates. After 48 h, cultures were rinsed 3 timeswith serum-free DMEM and 3% swine serum/DMEMwas added. After a 96-h incubation, cells were rinsed 3times with DMEM, and 3% HS/BSA-linoleic acid (1.5

g/mL)/DMEM was added. At 120 h, ZH (0, 0.0001,0.001, 0.01, and 1 M) in 3% HS/BSA-linoleic acid(1.5 g/mL)/DMEM was added. At 168 h, total RNAwas isolated using the Absolutely RNA Microprep Kit(Stratagene). The concentration of RNA and cDNAsynthesis was measured as described previously.

Real-Time Quantitative PCR

Real-time quantitative-PCR was used to measurethe quantity of 1-AR, 2-AR, and 3-AR and IGF-Igene expression relative to the quantity of 18S rRNAin total RNA isolated from bovine satellite cells treat-ed with ZH. Measurement of the relative quantity ofcDNA was performed using TaqMan Universal PCRMaster Mix (Applied Biosystems), 900 nMof the ap-propriate forward and reverse primers, 200 nMof ap-propriate TaqMan detection probe, and 1 L of thecDNA mixture. The bovine-specific 1-AR, 2-AR, and3-AR, IGF-I forward and reverse primers, and Taq-Man detection probes (Table 1) were designed usingpublished GenBank sequences. Commercially availableeukaryotic 18S rRNA primers and probes were used asan endogenous control (Applied Biosystems; GenBankaccession no. X03205). The ABI Prism 7000 detectionsystem (Applied Biosystems) was used to perform theassay using the thermal cycling variables recommendedby the manufacturer (50 cycles of 15 s at 95C and 1

min at 60C). The 18S rRNA endogenous control wasused to normalize the expression of 1-AR, 2-AR, and3-AR and IGF-I.

Preparation of Protein Extracts

Total protein was isolated using the mammalian pro-tein extraction reagent (Pierce Biotechnology, Rock-ford, IL). Bovine satellite cells were plated as describedpreviously on 9.62-cm2 collagen-coated plates. For myo-blast cultures, after a 144-h incubation, cells were rinsed3 times with serum-free DMEM and 10% FBS/DMEMwas added. For myotube cultures, after 144 h of incuba-

tion, cells were rinsed 3 times with serum-free DMEMand 3% HS/DMEM was added with ZH (0, 0.001, 0.01,0.1, 1, and 10 M). At 168 h, for myotubes, cells wererinsed with warm PBS and extraction reagent (350 L/well) was added. The cultures were gently shaken for 5min to ensure complete cell lysis and then centrifuged

Table 1. Sequences for -1, -2, and -3 adrenergic receptors and IGF-I specific PCR primers and TaqMan probes(Applied Biosystems, Foster City, CA)

Item Sequence

-1 adrenergic receptor (accession No. AF188187)

Forward GTGGGACCGCTGGGAGTATReverse TGACACACAGGGTCTCAATGCTaqMan probe 6FAM-CTCCTTCTTCTGCGAGCTCTGGACCTC-TAMRA

-2 adrenergic receptor (accession No. NM_174231)Forward CAGCTCCAGAAGATCGACAAATCReverse CTGCTCCACTTGACTGACGTTTTaqMan probe 6FAM-AGGGCCGCTTCCATGCCC-TAMRA

-3 adrenergic receptor (accession No. XF86961)Forward AGGCAACCTGCTGGTAATCGReverse GTCACGAACACGTTGGTCATGTaqMan probe 6FAM-CCCGGACGCCGAGACTCCAG-TAMRA

IGF-1 (accession No. X15726)Forward TGTGATTTCTTGAAGCAGGTGAAReverse AGCACAGGGCCAGATAGAAGAGTaqMan probe 6FAM-TGCCCATCACATCCTCCTCGCA-TAMRA

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for 5 min at 14,000 g. The supernatant was collect-ed and protein concentration was determined using aND-1000 Spectrophotometer (NanoDrop Technologies,Wilmington, DE). Average protein concentration col-lected was 8 mg/mL.

Western Blot Analysis

Protein samples were denatured using equal volumeof SDS--mercaptoethanol and boiled for 2 min. Totalprotein (30 g) was then separated by gel electropho-resis using Novex 1020% Tris-glycine gels (Invitrogen,Carlsbad, CA). Gels were run for 100 min at 125 Vand 130 mA. The protein was then transferred ontoa polyvinylidene fluoride membrane (Bio-Rad, Hercu-les, CA). The polyvinylidene fluoride membranes wereblocked using XCell SureLock Mini-Cell (Invitrogen)for 120 min at 4C. The primary antibody against 2-AR (ab40834, Abcam Inc., Cambridge, MA; Steenhuiset al., 2011) and GAPDH (ab9484, Abcam Inc.) inblocking buffer was added to the membrane and in-cubated overnight at 4C. After overnight incubation,the membrane was washed 3 times with PBS-Tween20. The secondary antibody for the 2-AR primaryantibody (ab6885; Abcam Inc., Cambridge, MA) andfor the glyceraldehyde 3-phosphate dehydrogenase pri-mary antibody (ab6789; Abcam Inc.) were added to themembrane and incubated for 2 h. Blots were washed 3times with PBS-Tween 20 and exposed to X-ray film(Carestream Health, Rochester, NY) for detection. Thepeak heights of 2-AR and glyceraldehyde 3-phosphatedehydrogenase bands were quantified using a Bio-RadImaging System (Bio-Rad).

Bovine Adipose Tissue Explant System

Two experiments were conducted to establish the ef-fects of ZH on de novo lipid synthesis. Subcutaneousadipose tissue from over the scapula was obtained atslaughter from finished steers that were being processedat the Texas A&M University Rosenthal Meat Scienceand Technology Center. Samples were obtained within20 min of exsanguination. Adipose tissue samples (50to 100 mg) were transferred to 25-mL flasks contain-ing 5 mMglucose, 5 mMacetate, 10 mMHEPES buf-fer, and 1 Ci [1-14C]acetate in Krebs-Henseleit bufferflasks (May et al., 1994) and 0, 1, 10, 100, or 1,000 MZH (n = 3 steers; trial 1) or 0, 1, 10, or 1,000 MZH(n = 3 steers; trial 2). Flasks in trial 2 also contained500 M theophylline to inhibit phosphodiesterase ac-tivity. Vials were gassed for 1 min with 95% O2:5%CO2 and incubated for 2 h in a shaking water bath at37C. At the end of the incubation period, reactionswere terminated by addition of 1 mL of 2 NH2SO4. Theneutral lipids in the adipose tissues were extracted bythe chloroform:methanol procedure (May et al., 1994),evaporated to dryness, and resuspended in 10 mL of

scintillation cocktail, and radioactivity was countedwith a scintillation counter.

Statistical Analysis

Data were analyzed as a completely randomized de-sign using PROC MIXED (SAS Inst. Inc., Cary, NC).The difference between control and treatment was eval-

uated using the least significance difference procedureof SAS.

RESULTS AND DISCUSSION

Effect of ZH on Cel l Proliferation,Differentiation, and mRNA Expressionof the -AR, and IGF-I mRNAin Bovine Satellite Cells

Satellite cells are mononucleated cells that are impor-tant in postnatal skeletal muscle growth (Mauro, 1961;

Moss and Leblond, 1971). Because of the postmitoticnature of the muscle fiber, the fusion of satellite cellswith muscle fibers is necessary to support postnatalmuscle growth. These cells are the external source ofDNA required for the muscle fiber to sustain musclehypertrophy, and this DNA accumulation is highly cor-related to muscle growth rate (Trenkle et al., 1978).Because of their role in postnatal muscle growth, sat-ellite cells are useful tools in investigating the modeof action of different muscle growth-promoting agents,such as -AA.

There was no effect of ZH on bovine satellite cellrate of proliferation as measured by [3H]thymidine in-

corporation (Figure 1). Additionally, ZH did not al-ter the extent of differentiation of bovine satellite cellsas assessed by percentage fusion (data not shown).OConnor et al. (1991b) reported a decrease in DNAconcentration in skeletal muscle of ram lambs fed ci-materol. The hind limb muscle DNA concentration wasreduced by 42% during a 3-wk of administration of ci-materol and remained 25% less after 6 wk of treatment.The total weight and protein content of the musclesfrom the hind limb of the lambs was increased by 30%during the 3-wk administration of cimaterol, suggestingthat an increase in satellite cell proliferation was notnecessary to support the -agonist induced muscle hy-pertrophy. Rehfeldt et al. (1997) observed no alterationin the DNA content of skeletal muscles of broiler chicksadministered clenbuterol for 3 wk. Similarly, cull beefcows administered ractopamine for 35 d before slaugh-ter showed no change in satellite cell numbers andmuscle fiber associated nuclei (Gonzalez et al., 2007).In contrast to our data, Grant et al. (1990) observedan increase in cell proliferation in muscle satellite cellsisolated from chick breast muscle with ractopaminetreatment; however, there was no effect on the fusion ofthose cells into the muscle fiber.

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6 wk, there was a decrease in circulating IGF-I concen-trations (Beermann, 1987). Conversely, clenbuterol ad-ministration to rats increased IGF-I mRNA and IGF-Icontent skeletal muscle without any change in serumIGF-I (Awede et al., 2002). This report is consistentwith the increase in IGF-I mRNA we observed.

There was no effect (P> 0.10) of ZH on the expres-sion of genes analyzed in fused myotube cultures at 192h (Figures 4 and 5). Shappell et al. (2000) reportedincreased cell number, protein, and DNA concentra-tions in C2C12 myoblasts after ractopamine treatment;however, no differences were reported in fused myotube

cultures treated with ractopamine. Thus, under ourculture conditions, ZH administration to bovine satel-lite cell myotube cultures had no effect on the expres-sion of the -AR and IGF-I mRNA in fused myotubes.

Effect of ZH and the 2-AR AntagonistICI-118, 551 on the mRNA Expressionof the -AR and IGF-I mRNAin Proliferating Bovine Satellite Cells

We used the specific 2-AR antagonist ICI-118, 551to determine whether the effects of ZH in myoblast

Figure 2. Zilpaterol (Merck Animal Health, Whitehouse Station, NJ) decreased myoblast A) 1-adrenergic receptor (AR; P= 0.07, n = 7),B) 2-AR (P< 0.05, n = 7), and C) 3-AR mRNA (0.01 M, P< 0.01; 1.0 M, P= 0.06, n = 7). Bovine satellite cells were plated in 10% fetalbovine serum/Dulbeccos modified Eagle medium. Zilpaterol was added at 48 h. Total RNA was isolated at 96 h, and relative mRNA abundancewas determined by real-time PCR. Data points represented as relative to control. Bars without a common letter differ (P= 0.07). Bars are meanvalues relative to control SEM.

Figure 3. Zilpaterol (Merck Animal Health, Whitehouse Station,NJ) increased myoblast IGF-I mRNA (P= 0.07, n = 7). Bovine satel-lite cells were plated in 10% fetal bovine serum/Dulbeccos modifiedEagle medium. Zilpaterol was added at 48 h. Total RNA was isolatedat 96 h, and relative mRNA abundance was determined by real-timePCR. Data points represented as relative to control. Bars withouta common letter differ (P= 0.07). Bars are mean values relative tocontrol SEM.

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cultures were mediated through the 2-AR. The 2-ARantagonist ICI-118, 551 at 1 Mattenuated the reducedexpression of 1-AR mRNA elicited by 0.01 M ZH(Figure 6A). The combination of these concentrationsof IC-118, 551 and ZH increased (P < 0.05) 1-ARgene expression. For the expression of 2-AR, both 0.01Mand 0.1 M ICI-118,551 blocked the reduction inmRNA caused by 0.01 MZH (Figure 6B). There wasno effect (P> 0.10) of IC-118, 551 on 3-AR mRNA,nor was there an effect (P> 0.10) of ZH (Figure 6C).There also was no effect (P> 0.10) of ICI-118, 551alone or in combination with ZH on the expression ofIGF-I mRNA (Figure 7). The ICI-118, 551 was used in

this culture system to determine whether the effects onthe expression of the receptors and IGF-I mRNA wasmediated through the 2-AR. In a previous study, ICI-118551 blocked the effect of ZH in lipopolysaccharide-exposed u937 macrophages (Verhoeckx et al., 2005).In a similar manner, ractopamine-stimulated increasesin cell number, protein, and DNA concentrations wereattenuated by propranolol, an antagonist for both the1- and 2-AR (Shappell et al., 2000). The stimulationof lipogenesis by ractopamine in rat adipocytes waspartially inhibited by propranolol as well (Hausman etal., 1989). In our study, the results indicate that thereduction in -AR mRNA by ZH was mediated through

Figure 4. Effect of zilpaterol (Merck Animal Health, Whitehouse Station, NJ) on myotube A) 1-adrenergic receptor (AR), B) 2-AR, andC) 3-AR mRNA. Bovine satellite cells were plated in 10% fetal bovine serum/Dulbeccos modified Eagle medium. After 24 h, 3% swine serum/Dulbeccos modified Eagle medium was added, and 3% horse serum/Dulbeccos modified Eagle medium was added at 144 h. Zilpaterol was addedat 168 h to fused myotubes. Total RNA was isolated at 216 h, and relative mRNA abundance was determined by real-time PCR. Data pointsrepresented as relative to control. Bars are mean values relative to control SEM. There was no effect (P> 0.10) of treatment on the expressionof 1, 2, and 3-AR mRNA (n = 5).

Figure 5. Effect of zilpaterol (Merck Animal Health, WhitehouseStation, NJ) on myotube IGF-I mRNA. Bovine satellite cells wereplated in 10% fetal bovine serum/Dulbeccos modified Eagle medium.After 24 h, 3% swine serum/Dulbeccos modified Eagle medium wasadded, and 3% horse serum/Dulbeccos modified Eagle medium wasadded at 14 h. Zilpaterol was added at 168 h to fused myotubes.Total RNA was isolated at 216 h and relative mRNA abundance wasdetermined by real-time PCR. Data points represented as relative tocontrol. Bars are mean values relative to control SEM. There wasno effect (P> 0.10) of treatment on the expression of IGF-I mRNA(n = 5).



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the 2-AR because of the ability of ICI-118,551 to ame-liorate these effects.

Effect of ZH on Bovine Satellite Celland 2-AR Protein Expression

Western blot analysis revealed that the protein con-tent of 2-AR in ZH-treated myotube cultures de-creased (P= 0.05) relative to control (Figure 8) Wewere unable to detect differences in the mRNA expres-sion of 2-AR mRNA in myotube cultures, but therewas an decrease in protein in the Western blot analysisfor 2-AR. This indicated that the decrease in 2-ARprotein expression due to ZH treatment may be in re-sponse to a posttranscriptional event. The reduction inprotein expression from myotubes supports much of theresearch suggesting -agonists decrease receptor densi-ty (Spurlock et al., 1994; Huang et al., 2000; Walker etal., 2007). In contrast, ractopamine administration hasled to a tendency for an increase in 2-AR mRNA inskeletal muscle isolated from yearling steers and feedlot

heifers collected at slaughter (Sissom et al., 2007; Win-terholler et al., 2007). Also, in chicken skeletal musclecells treated with isoproterenol (a -agonist), there wasan increase in -AR population of 40% between d 7 and10 in culture (Young et al., 2000).

Lipogenesis in ZH-Treated AdiposeTissue Explants

Because of the relative insensitivity of bovine adi-pose tissue to -AA, including epinephrine (Prior et al.,1983; Miller et al., 1989), greater concentrations of ZHwere used in the experiments with adipose tissue ex-plants than in those using satellite cell cultures. In thefirst adipose tissue trial, in which theophylline was notincluded in incubation media, ZH stimulated acetateincorporation into total lipids (Table 2). Similarly, fattyacid synthesis was stimulated by ractopamine in ratadipocytes; this was partially inhibited by propranolol,indicating that the stimulation of lipogenesis by rac-topamine involved the -AR (Hausman et al., 1989).

Figure 6. Zilpaterol decreased and zilpaterol (Merck Animal Health, Whitehouse Station, NJ) + ICI-118, 551 combination increased myoblast1-adrenergic receptor (AR) mRNA (A: P< 0.05, n = 4). Additionally, zilpaterol decreased myoblast 2-AR mRNA, and ICI-118, 551 blockedthe reduction (B: P< 0.05, n = 4) and had no effect on 3-AR mRNA (C). Bovine satellite cells were plated in 10% fetal bovine serum/Dul-

beccos modified Eagle medium. Zilpaterol was added at 48 h. Total RNA was isolated at 96 h, and relative mRNA abundance was determinedby real-time PCR. Data points represented as relative to control. Bars without a common letter differ (P< 0.05). Bars are mean values relativeto control SEM.

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Adenosine accumulates during adrenergic stimulationof adipose tissue, which causes inhibition of adenylatecyclase (Fredholm, 1981; Mills and Mersmann, 1995).Theophylline enhances the action of -AA in adiposetissue by inhibiting phospodiesterase (Arner et al.,

1993). This increases the concentration of cyclic ad-enosine monophosphate and can overcome the effectsof adenosine. Therefore, theophylline was included inthe media for the second trial. With the addition of500 Mtheophylline, ZH caused a dose-dependent re-duction in lipid synthesis from acetate when data wereexpressed as a percentage of control values (Table 2).However, even at the greatest concentration of ZH, li-pogenesis from acetate was depressed only about 40%.

In a previous study (Miller et al., 1988), we were unableto demonstrate an effect of 50 Mepinephrine plus 500Mtheophylline on acetate incorporation into lipids inbovine s.c. adipose tissue, even though adipose tissuesfrom clenbuterol-fed steers exhibited strongly depressedrates of lipogenesis in vitro. Miller et al. (1989) testedthe effects of clenbuterol in short-term incubations of

Figure 7. Effect of zilpaterol (Merck Animal Health, WhitehouseStation, NJ) and ICI-118, 551 alone or in combination on myoblastIGF-I mRNA. Bovine satellite cells were plated in 10% fetal bovineserum/Dulbeccos modified Eagle medium. Zilpaterol and ICI-118,551 was added at 48 h. Total RNA was isolated at 96 h, and relativemRNA abundance was determined by real-time PCR. Data pointsrepresented as relative to control. Bars are mean values relative tocontrol SEM (n = 4).

Figure 8. A) Western blot image of zilpaterol (Merck AnimalHealth, Whitehouse Station, NJ)-induced decrease in 2-adrenergicreceptor (AR) in myotube cultures. B) Zilpaterol decreased 2-ARprotein expression in myotube cultures (P < 0.05, n = 4). Bovinesatellite cells were plated in 10% fetal bovine serum/Dulbeccos modi-fied Eagle medium for 144 h. Myotubes were cultured with 3% horseserum/Dulbeccos modified Eagle medium with or without zilpaterolfor 168 h. Total protein was isolated and used in Western blot analysis.Bars are mean values SEM.

Table 2. Total lipid synthesis from acetate in subcutaneous adipose tissue incubated with zilpaterol hydrochloride(Merck Animal Health, Whitehouse Station, NJ)

Item1

Zilpaterol hydrochloride, M

SEM P-valueControl 1 10 100 1,000

Trial 1Rate2 8.4 24.1 21.4 27.4 28.6 5.6 0.16Percentage control 243 232 337 352 33.6 0.004

Trial 2Rate 16.2 15.3 13.3 7.2 4.4 0.25Percentage control 71 51 41 13 0.05

1Trial 1: acetate conversion to lipids in subcutaneous (s.c.) adipose tissue, no theophylline. Trial 2: acetate conversion to lipids in s.c. adiposetissue, 500 Mtheophylline.

2Acetate (nmol) incorporated into total lipids/(2 h/100 mg of adipose tissue). All values are means for 3 steers (lipid synthesis) or preadipocytecultures (SCD gene expression).



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bovine s.c. adipose tissue from control cattle and fromthe cattle treated with 7 mg clenbuterol/heifer per dayfor 50 d. Lipogenesis from acetate was not affected byclenbuterol in control steers (835 vs. 604 nmol2 h1105cells1) or clenbuterol-treated steers (413 vs. 383 nmol2h1105 cells1).

Overall Conclusions

These data suggest that the expression of 2-AR wasdifferentially regulated by ZH in myoblasts and myo-tubes. Additionally, they would support previous re-search suggesting that the expression of -AR in bovineskeletal muscle cells can vary due to variables such astreatment with -agonists and time in culture (Bridgeet al., 1998). These data further support the impor-tant role the 2-AR plays in modulating the functionof ZH on skeletal muscle growth. Finally, the minimaleffects of ZH on de novo fatty acid biosynthesis in ourbovine adipose tissue explant system are consistentwith the previously reported, modest effects of other

-adrenergic agents on adipose tissue metabolism.Thus, the reduction in carcass adiposity frequently (al-though not invariably) observed in cattle treated with-AA more likely is attributable to redirection of nu-trients from adipose tissue fatty acid biosynthesis tomuscle accretion.

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