J. Anim. Sci. 2003. 81:662-668
© 2003 American Society of Animal Science
ß-Adrenergic receptor subtypes that mediate ractopaminestimulation of lipolysis1,2
S. E. Mills*,3,
M. E. Spurlock* and
D. J. Smith
* Department of Animal Sciences, Purdue University, West Lafayette, IN 47907 and
and
USDA, ARS, Biosciences Research Laboratory, Fargo, ND 58105
3 Correspondence: Lilly Hall (phone: 765-494-4845; fax 765-494-9346; E-mail:smills{at}purdue.edu).
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Abstract
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Ractopamine HCl is an ß-adrenergic receptor (ßAR) ligand that was recently approved for use in swine to enhance carcass leanness. The RR stereoisomer of ractopamine is the most active of the four stereoisomers exhibiting the highest affinity and signaling response. The RR isomer exhibits selective activation of the porcine ß2AR, which might limit the lipolytic response to ractopamine because the ß1AR is the predominant subtype in swine adipocytes and may mediate most of the lipolytic response. Therefore, we determined the ßAR subtypes that mediate the lipolytic response to ractopamine in swine adipocytes. In order to confirm the predominant role of the ß1AR in porcine adipocytes, isoproterenol-stimulated lipolysis was inhibited by increasing doses of subtype-selective antagonists. Inhibition curves were biphasic using ß1AR antagonists (CGP 20712A and bisoprolol) and curve analysis indicated that both ß1AR and ß2AR contributed to lipolysis with 50 to 60% of the response coming from the ß1AR. Inhibition with the ß2AR antagonist clenbuterol revealed only one class of ßAR that closely approximated the kinetics of the ß1AR. When the RR isomer of ractopamine was the lipolytic agent, similar results to isoproterenol were observed, except that the estimated contribution of the ß1AR was 38%. That ß2AR antagonists did not detect a contribution of the ß2AR to lipolysis may indicate that the ß1AR masked the response to the ß2AR. Dose titration with the RR isomer in the presence of a saturating concentration of ß1AR or ß2AR antagonists indicated that each subtype was present in sufficient quantities to stimulate lipolysis near maximally. Data indicate that both the ß1AR and ß2AR are functionally linked to lipolysis in swine adipocytes and that ractopamine activates each subtype. The RR isomer of ractopamine stimulated adenosine 3',5'-cyclic phosphate accumulation with equal efficacy to isoproterenol through the cloned porcine ß2AR, but was only 35% as efficacious through the cloned porcine ß1AR. These data confirm the ß2AR selectivity of the RR stereoisomer, but suggest the partial agonism through the ß1AR is sufficient to activate lipolysis through both subtypes in swine adipocytes.
Key Words: ß-Adrenergic Receptors • Lipolysis • Pigs
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Introduction
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Triglyceride hydrolysis in adipocytes is regulated by catecholamines that bind to ß-adrenergic receptors (ßAR) to activate hormone-sensitive lipase (Fain and Garcia-Sainz, 1983). Adipocytes express three ßAR subtypes, but the role for each subtype is not clearly defined and may differ among species. For rodents, the ß3AR is the predominant subtype and mediates the majority of the lipolytic response (Hollenga and Zaagsma, 1989). In the pig, the ß1AR represents nearly 80% of the total receptors (McNeel and Mersmann, 1999; Liang and Mills, 2002) and seems to be the primary subtype mediating lipolysis. The ß2AR may be uncoupled from the lipolytic cascade, a conclusion based on the finding that the ß2AR-selective ligand BRL-37344 did not interfere with isoproterenol-stimulated lipolysis at low concentrations (ß2AR-mediated), but did at high concentrations (ß1AR-mediated; Mills, 2000). Because this conclusion was based on data using only one selective ligand, it was of interest to confirm these results with additional subtype-selective ligands.
Ractopamine is a phenethanolamine ßAR agonist that promotes muscle growth in swine (Moody et al., 2000). The commercial product is a mixture of four stereoisomers, of which, one (RR) seems to be the functional compound (Ricke et al., 1999; Mills et al., 2002). Ractopamine binds porcine ßAR and stimulates lipolysis in vitro (Liu et al., 1989; Spurlock et al., 1993b). It is not clear which ßAR mediates the response to ractopamine. For species other than swine, ractopamine is suggested to have some selectivity for the ß1AR (Smith et al., 1990; Moody et al., 2000). For the pig, the ß2AR provides the best signal transduction (Mills et al., 2002), whereas binding affinity is similar for the ß1AR and ß2AR (Spurlock et al., 1993b; Liang et al., 2000; Mills et al., 2002). It was of interest, therefore, to determine which ßAR mediates the activation of lipolysis in swine adipocytes.
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Materials and Methods
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Materials
Ractopamine—[1R*,3R*], [1 R * , 3 S *] - 4 - hydroxy - 
-([{ 3 - (4 - hydroxy [1 4C] phenyl) - 1 - methylpropyl } amino]methyl) - benzenemethanolhydrochloride —stereoisomers were synthesized as described by Ricke et al. (1999). Other drugs were purchased from the following companies: clenbuterol and (-)isoproterenol from Sigma Chemical (St. Louis, MO), bisoprolol from TOCRIS (Ballwin, MO), and GCP20712A from RBI (Natick, MA).
Adipocytes and Lipolysis
The middle layer of subcutaneous adipose tissue (fourth to 10th ribs) was taken at the time of death from market-weight barrows (PIC line 337 sires x York-Landrace dams). Pigs were killed by exsanguination following electrical stunning at the Purdue University abattoir. Adipocytes were isolated by collagenase digestion, and cell number was determined from the average cell size as measured from osmium tetroxide-fixed cells and total lipid (Liu et al., 1989). Adipocytes (approximately 105 cells/mL) were washed and suspended in incubation buffer (Krebs-Ringer bicarbonate [KRB] containing 1.25 mM CaCl2, 0.5 mM ascorbic acid, 10 mM HEPES, 5 mM glucose, and 3% BSA, pH 7.4, as described by Liu et al. (1989). To quantify rates of lipolysis, duplicate 0.5-mL aliquots of the cell suspension were incubated in 17 x 100 mm polyethylene tubes in an atmosphere of 5% CO2 in oxygen. Tubes contained theophylline (0.4 mM) and ßAR ligands as specified in Figures 1 through 4
. Vials were shaken in a gyratory water bath at 37°C for 2 h. Incubations were stopped by adding 0.025 mL of 35% (vol/vol) HCLO4, and glycerol was quantified in neutralized, protein-free extracts using a commercial kit adapted for 96-well plates (GPO-Trinder triglyceride kit; Sigma Chemical). Lipolytic rates were expressed as nmoles glycerol released min-1•106•cells-1.
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Figure 1. Effect of dose on stimulation of lipolysis in porcine adipocytes by isoproterenol and inhibition of isoproterenol (10-7 M) stimulated lipolysis by antagonists selective for ß-adrenergic receptor subtypes. Data are representative of three independent experiments with different pigs. Curves were fit by nonlinear regression and results are summarized in Table 1 .
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Figure 2. Effect of dose on stimulation of lipolysis in porcine adipocytes by the RR stereoisomer of ractopamine and inhibition of RR (10-7 M) stimulated lipolysis by antagonists selective for ß-adrenergic receptor subtypes. Data are representative of two independent experiments with different pigs. Curves were fit by nonlinear regression and results are summarized in Table 2.
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Figure 3. Effect of dose on stimulation of lipolysis in porcine adipocytes by the RR stereoisomer of ractopamine in the absence and presence of antagonist for the ß1-adrenergic receptor (CGP 20712A; CGP) or the ß2-adrenergic receptor (BRL 37344; BRL). Data are representative of three independent experiments with different pigs. Curves were fit by nonlinear regression, and results are presented in Table 3. The concentrations of CGP (50 nM) and BRL (2 or 5 µM) were selected to block the ß1 or ß2AR adrenergic receptor respectively without interference of the other subtype and were determined from the affinity of each ligand and the concentration of RR according to the equation of Cheng and Prusoff (1973).
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Figure 4. Activation of adenylyl cyclase by isoproterenol or the RR stereoisomer of ractopamine through the porcine ß1- and ß2-adrenergic receptor expressed in Chinese hamster ovary cells. Intact cells were incubated for 40 min in the absence or presence of ligand (10-4 M), and adenosine 3',5'-cyclic phosphate was quantified by radioimmunoassay. Data are the means ± SEM for three independent experiments with cloned cells grown on different dates.
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Cell Lines
Stably transfected Chinese hamster ovary cell lines expressing the porcine ß1AR or ß2AR were grown in an atmosphere of 95% air and 5% (CO2 at 37°C in a 1:1 F12:Dulbecco’s modified Eagle’s medium DMEM) media containing 100 U/mL of ampicillin, 200 U/mL of penicillin, 200 µg/mL of streptomycin, 10-8 M Se, and 1.2 mg/mL of NaHCO3 in 1.5 mM HEPES, pH 7.4), plus 10% FBS and G418 (0.5 mg/mL; Liang et al., 2000). Confluent cells were washed twice with warm F12:DMEM and detached with 0.4% trypsin in F12:DMEM. Cells were centrifuged at 300 x g at 4°C for 5 min and washed twice in KRB media containing 0.1% BSA. Whole cells were suspended in the same media and 0.1 mL was distributed to 1.5-mL microcentrifuge tubes for determination of adenylyl cyclase activation. Tubes contained theophylline (0.4 mM) and test ligands in a final volume of 0.15 mL and were incubated for 40 min at 37°C. Incubations were stopped with 1 N NaOH and neutralized with 1 M acetic acid containing universal indicator (Sigma Chemical). Neutralized extracts were used for the assay of adenosine 3',5'-cyclic phosphate (cAMP) by RIA using the protocol supplied with the cAMP antibody (Calbiochem, San Diego, CA). Samples and standards were acetylated to increase the sensitivity of the assay. A portion of the cell suspension was used to determine cell number by determination of DNA content. Cells were sonicated for 10 s to disrupt the cell structure. DNA was quantified using the fluorescent dye H 33258 (Sigma Chemical) according to Labarca and Paigen (1980). Data were expressed as fentamoles of cAMP per 106 cells.
Data were analyzed using the GLM procedures of SAS for a completely randomized design (SAS Inst., Inc., Cary, NC). Lipolytic response curves were analyzed for best-fit using nonlinear regression analysis and kinetic parameters were determined (Prism, GraphPad Software Inc., San Diego, CA). Lipolysis studies were conducted using adipocyte preparations from two or three pigs. The effect of ßAR subtype inhibitors on kinetic parameters was determined using single degree of freedom orthogonal contrasts for differences from values in the absence of drugs. In order to determine the activation of adenylyl cyclase in cultured cells, cells were maintained in culture and cells propogated for each experiment. Replicate experiments represented cells plated at different times.
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Results
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The ß1AR and ß2AR comprise nearly 95% of the ßAR in swine adipocytes (McNeel and Mersmann, 1999), so determination of the contribution of these two subtypes to lipolysis will likely account for essentially all of the lipolytic response. To determine the contribution of the ß1AR and ß2AR to lipolysis, subtype-selective ligands were used to inhibit lipolysis stimulated by the nonselective agonist isoproterenol. As shown in Figure 1, isoproterenol stimulated lipolysis in a dose-response fashion with a half-maximal dose of approximately 10-8 M. Based on this dose response, subsequent experiments were conducted using 10-7 M isoproterenol to achieve a near maximal rate. The plots for the competitive inhibition of isoproterenol-stimulated lipolysis by two ß1AR-selective antagonists, CGP 20712A and bisoprolol (400- and 33-fold selective for the pß1AR; Cao, 1998; Liang and Mills, 2001) and the ß2AR-selective ligand clenbuterol (25-fold selective for the pß2AR; Cao, 1998; Liang and Mills, 2001) are shown in Figure 1. Both ß1AR antagonists exhibited biphasic displacement curves indicative of the titration of two ßAR subtypes. Nonlinear analysis of competition curves yielded affinity estimates for the high- and low-affinity sites that were reasonably close to expected values for the porcine ß1AR and ß2AR (Table 1). Values for the inhibition constant, Ki, for the high- and low-affinity sites, respectively, were 1.6 and 2,630 nM with CGP 20712A as competitor, and 13.6 and 807 nM with bisoprolol as competitor. Expected values are based on data for each drug using the cloned ß1AR and ß2AR (Cao, 1998; Liang et al., 2000). Using ß1AR selective antagonists, these data suggest that both the ß1AR and ß2AR contribute to the lipolytic response stimulated by isoproterenol. The percentage contribution of each receptor subtype was also determined from the kinetic analysis and indicated that approximately 50% of the response was contributed by each subtype (Table 1). Clenbuterol is a partial agonist toward porcine ßAR (Liu et al., 1989; Spurlock et al., 1993b), and under the conditions of this assay, clenbuterol is a functional antagonist. Inhibition by clenbuterol did not reveal two classes of receptors as for the ß1AR antagonist and the inhibition curves modeled best to one site (Figure 1). The calculated Ki of 430 nM was closer to the value for the ß1AR (300 nM) than to the ß2AR (10 nM), suggesting that the ß1AR was contributing to the observed response (Table 1). Thus, a different picture emerges when a ß1AR or ß2AR antagonist is used.
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Table 1. Estimates of binding affinity for adenosine ß-adrenergic receptor ligands obtained from competitive inhibition of isoproterenolstimulated lipolysisa
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To determine which ßAR mediates the lipolytic response to the active stereoisomer of ractopamine, competitive inhibition experiments were conducted using the RR stereoisomer with ß1AR and ß2AR antagonists. The ß1AR-selective ligand CGP 20712A yielded a two-component inhibition curve (Figure 2). Kinetic analysis of these curves showed affinity estimates that were reasonably close to the expected values for ß1AR and ß2AR and a contribution by the ß1AR of 38% (Table 2). Once again, clenbuterol yielded a single-component displacement curve with an estimated Ki of 262 nM, which is close to the dissociation constants Kd of clenbuterol for the porcine ß1AR. Results are similar to the data when using isoproterenol as the agonist and indicate that both the ß1AR and ß2AR contribute to the stimulation of lipolysis under conditions of a high concentration of the RR stereoisomer of ractopamine.
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Table 2. Estimates of binding affinity for ß-adrenergic receptor ligands obtained from competitive inhibition of ractopamine-stimulated lipolysisa
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To examine the functional activity of each ßAR subtype over a range of ractopamine concentrations, dose response experiments were conducted with the RR stereoisomer in the presence of concentrations of CGP 20712A (50 nM) and BRL 37344 (2,000 or 5,000 nM) calculated to block RR binding to the ß1AR or ß2AR respectively. We substituted BRL 37344 for clenbuterol because this ß2AR ligand has a greater selectivity for the ß2AR (75-fold) than has clenbuterol (Liang et al., 2000). The RR isomer stimulated lipolysis in a dose-dependent manner in the presence of antagonist, confirming that both subtypes contribute to the lipolytic response (Figure 3). Both antagonists shifted the dose response curve to the right. The shift in EC50 (the concentration of ligand that results in half-maximal stimulation) was greater when the ß1AR was blocked (sixfold) than when the ß2AR was blocked (threefold), indicating a greater contribution of the ß1AR (Table 3). The presence of either antagonist prevented full lipolytic expression at RR stereoisomer concentrations up to 10-6 M. The fact that a near-full lipolytic response is achieved through the ß2AR (+CGP 20212A) suggests that each subtype is present in sufficient quantities to regulate lipolysis in the pig.
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Table 3. Kinetic variables for the stimulation of lipolysis by ractopamine through the ß1- or ß2-adrenergic receptora
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We have shown previously that the RR isomer of ractopamine activated adenylyl cyclase more efficiently through the ß2AR than the ß1AR using membranes from cell lines expressing each receptor subtype (Mills et al., 2002). In fact, adenylyl cyclase activation was undetectable when ß1AR were used. These results differ from the present findings in that the RR isomer stimulated lipolysis through both the ß1AR and ß2AR. To clarify whether RR activates the cloned ß1AR, we quantified cAMP accumulation in intact Chinese ovary cells that express either the ß1AR or ß2AR. Using intact cells, the RR isomer stimulated cAMP accumulation through either the ß1AR or ß2AR (Figure 4). The RR isomer was more efficacious through the ß2AR, however, being equal to isoproterenol, but was only about 35% as effective as isoproterenol through the ß1AR. These data indicate that RR may be a full agonist through the ß2AR but a partial agonist through the ß1AR.
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Discussion
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Adipocytes of all species studied to date express the three family members of ßAR, although the relative expression of each subtype differs considerably across species (Strosberg, 1990; Lafontan and Berlan, 1993). The coexistence of multiple ßAR suggests that a unique role exist for each subtype, but clearly distinct roles have not been identified. All three ßAR subtypes share a common signaling pathway through activation of adenylyl cyclase so that the greatest differences between subtypes seem to be in the relative level of expression, regulation of gene expression, and binding and signaling kinetics to natural and synthetic ligands (Granneman, 1995). The predominant ßAR expressed in porcine adipocytes is the ß1AR (McNeel and Mersmann, 1999; Liang and Mills, 2002). We have presented evidence that the ß1AR is the primary regulator of lipolysis in the pig and that the ß2AR may not be linked to the lipolytic cascade (Mills, 2000). These conclusions were based on data using the ß2AR-selective antagonist BRL 37344, which were very similar to the data reported herein with clenbuterol. With both drugs, the titration curve for the inhibition of lipolysis modeled to a single class of receptors with a calculated affinity similar to the ß1AR. We interpreted these data to suggest that the ß2AR did not participate in the stimulation of lipolysis. We further speculated that intracellular compartmentalization might uncouple the ß2AR from lipolysis. Evidence for a difference in the coupling efficiency of different ßAR subtypes, perhaps due to compartmentalization, has been reported (Hollenga et al., 1991). In their work, ligands that activated the ß3AR had a 10-fold greater coupling efficiency (rate of lipolysis/cAMP concentration) than did the nonselective agonist isoproterenol. Alternatively, Minneman and colleagues have suggested that ßAR subtypes do not function independently and that one subtype may prevail over the others depending on conditions (Zhong et al., 1996).
In contrast to our earlier conclusion, the present data do not provide evidence for compartmentalization and clearly demonstrate that both the ß1AR and ß2AR are functionally linked to lipolysis in the pig adipocyte. The participation of both ßAR subtypes was apparent when inhibiting isoproterenol-stimulated lipolysis with ß1AR antagonists, but not ß2AR antagonists (clenbuterol in this study and BRL 37344 in Mills, 2000). Why the ß2AR antagonist did not reveal two ßAR subtypes contributing to lipolysis is not clear. It does not appear to be due to low selectivity by the ß2AR antagonists because clenbuterol and BRL 37344 have a 30- to 75-fold selectivity for the ß2AR, whereas bisoprolol and CGP 20712A have a 50- to 400-fold selectivity for the ß1AR (Cao, 1998; Liang et al., 2000). One explanation may be that the ß2AR response is masked when the ß1AR is maximally activated and that inhibition by clenbuterol does not reduce lipolysis until the ß1AR is titrated. It should be pointed out that we were measuring a response to the binding of ligand and not binding itself, so not all ßAR may be accounted for if ßAR are in excess of the requirements for maximal lipolysis. The number of ßAR are typically in excess of what is required to stimulate lipolysis, and binding of only a small percentage of the total is sufficient to stimulate lipolysis maximally (Arner et al., 1976). Therefore, it is reasonable that because the ß1AR is the predominant subtype that this receptor alone could stimulate lipolysis maximally. Data in Figure 1 would suggest that the number of ß2AR is insufficient for maximal lipolysis. However, data from Figure 3 indicates that the ß1AR and ß2AR are equally efficacious in stimulating lipolysis. An alternative explanation may be that under conditions of maximal lipolysis, the ß1AR is the primary contributing subtype and that only when the number of functional ß1AR is reduced with an antagonist is a ß2AR component observed.
Analysis of the inhibition curves with isoproterenol as an agonist indicated that the ß1AR contributed 50 to 60% of the total lipolytic response. This value is less than the predicted value of 75% based on messenger RNA abundance (McNeel and Mersmann, 1999) or receptor number (Liang and Mills, 2002). Although we are not certain of the percentage of each ßAR subtype in the pigs used in the current studies, it is possible we have underestimated the contribution of the ß1AR because of spare receptors. It is possible that both CGP 20712A and bisoprolol reduced the number of functional ß1AR before a reduction in the rate of lipolysis was observed. If true, the contribution of ß1AR would have been underestimated. Alternatively, we cannot rule out the possibility that with collagenase digestion the number and/or ratio of ßAR may be altered, although we have observed greater ßAR density in membrane preparations from adipocytes than from adipose tissue (Spurlock et al., 1993a).
The commercial preparation of ractopamine is a mixture of four stereoisomers that result from the presence of two chiral carbons (Colbert et al., 1991; Smith, 1998). The RR isomer seems to be the active isomer because it has the highest affinity and greatest efficacy for adenylyl cyclase activation for pig ßAR (Mills et al., 2002), and because it can account for the growth response in rodents (Ricke et al., 1999). Ractopamine is reported to have ß1AR selectivity (Smith et al., 1990; Moody et al., 2000), but these data are based on rodent models and differ from data in the pig. The RR isomer has equal affinity for the porcine ß1AR and ß2AR, but more effectively couples to adenylyl cyclase through the ß2AR (Mills et al., 2002). Using membrane preparations from CHO cells expressing each subtype, the RR isomer was a partial agonist through the ß2AR, but no response was observed through the ß1AR (Mills et al., 2002). Results from the present study confirm and refine the previous observation by demonstrating that the RR isomer does signal better through the ß2AR. Using the more physiological assay of cAMP accumulation in intact cells, the RR isomer is shown to be a partial agonist through the ß1AR and a full agonist through the ß2AR (Figure 4). The same pattern of preferential activation of the ß2AR by ractopamine in rodent tissues was reported by Colbert et al. (1991).
Despite the RR isomer having only partial agonist activity through the ß1AR, both the ß1AR and ß2AR contributed to the stimulation of lipolysis by RR. Unlike isoproterenol, however, stimulation of lipolysis by the RR stereoisomer of ractopamine was mediated predominantly through the ß2AR (63%) rather than the ß1AR. Again, the contribution of the ß1AR may be underestimated in these experiments. One consequence of RR being only a partial agonist through the ß1AR is that a full agonist response may not be realized, particularly in adipose tissue where the ß1AR predominates. Although ractopamine is acutely lipolytic in the pig (Veenhuizen et al., 1987), we have demonstrated that responses appear to be lost quickly and that, in some cases, chronic feeding of ractopamine has no apparent effect on the metabolism or rate of accretion of adipose tissue (Dunshea, 1993; Liu et al., 1994). It is possible that a ligand with full agonist activity through the ß1AR would have an enhanced capacity to reduce lipid accretion in adipose tissue.
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Implications
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It is not clear why cells express more than one subtype of ß-adrenergic receptor, but the existence of multiple subtypes provides an opportunity to target the tissues and processes that are most desirable while eliminating the less desirable responses. Previous data suggested that ractopamine might preferentially target the ß2-adrenergic receptor, which may not be linked to lipolysis, and therefore may have limited effectiveness to reduce fat accretion in growing pigs. We report here that the ß2-adrenergic receptor is linked to lipolysis, and despite the fact that ractopamine is selective for the ß2-adrenergic receptor, the RR isomer can stimulate lipolysis through both the ß1-adrenergic receptor and ß2-adrenergic receptor. The ß1-adrenergic receptor may be the preferred target because it is the most abundant subtype in swine adipocytes, but targeting the ß2-adrenergic receptor should also result in reduced fat accretion in swine.
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Footnotes
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1 Journal paper No. 16865 of the Purdue University Agric. Res. Prog. 
2 Mention of trademark or proprietary product does not constitute a guarantee or warranty of the product by the USDA and does not imply its approval to the exclusion of other products that may also be suitable.
Received for publication August 7, 2002.
Accepted for publication October 10, 2002.
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Effects of a "step-up" ractopamine feeding program, sex, and social rank on growth performance, hoof lesions, and Enterobacteriaceae shedding in finishing pigs
J Anim Sci,
January 1, 2009;
87(1):
304 - 313.
[Abstract]
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J. M. Gonzalez, R. D. Dijkhuis, D. D. Johnson, J. N. Carter, and S. E. Johnson
Differential response of cull cow muscles to the hypertrophic actions of ractopamine-hydrogen chloride
J Anim Sci,
December 1, 2008;
86(12):
3568 - 3574.
[Abstract]
[Full Text]
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Top
Abstract
Introduction
