Insulin binding to mouse adipocytes exposed to clenbuterol and ractopamine in vitro and in vivo

DOMESTIC ANIMAL ENDOCRINOLOGY

Vol. 7(1):103-109, 1990

INSULIN BINDING TO MOUSE ADIPOCYTES EXPOSED TO CLENBUTEROL AND RACTOPAMINE IN VITRO AND IN VIVO 1 L.C. Dubrovin, C.Y. Liu and S.E. Mills 2`a Department of Animal Science Lilly Hall, Purdue University West Lafayette, IN 47907 Received July 28, 1989

ABSTRACT Insulin binding to mouse adipocytes was measured after in vitro (30 rain) and in vivo (5 days) exposure to clenbuterol and ractopamine. At 10"6M,both agonists decreased insulin binding by 20-30% after a 30 rain preincubation at each insulin concentration between 1 and 25 ng/ml. Binding was not decreased if propranolol was present. Scatchard plots suggested that decreased binding was due to a decrease in insulin receptor concentration. Insulin binding was decreased approximately 10% at agonist concentrations as low as 10 "t3 M, but binding was not further decreased until concentrations exceeded 10 "9 M. Rate of gain was increased 2-fold by clenbuterol (10 rag/liter of drinking water) and 50% by 500 mg ractopamine/liter, but not by 50 mg racroparnine/ liter. Clenbuterol and ractopamine (500 rag/liter) decreased fat pad weight but only clenbuterol increased hind limb muscle mass. Insulin binding following in vivo administration was not influenced by ractopamine at 50 rag/liter, but tended to be increased by clenbuterol and ractopamine at 500 mg/liter. The disparity in results between administering the ~-agonists in vitro or in vivo suggests that counter regulatory factors influenced insulin binding capacity in vtvo. Results indicate that ractopamine and clenbuterol can decrease insulin binding to adipocytes but the relevance of this response to decreased fat accretion is not clear.

INTRODUCTION When fed to livestock, clenbuterol (1,2), ractopamine (3,4) and other betaadrenergic agonists (5,6) have been shown to increase carcass protein and decrease carcass fat. Similar results were shown for clenbuterol in rodents (7). I n vitro, clenbuterol and other beta-adrenergic agonists induce insulin resistance in adipose tissue (7,8). Resistance to insulin is defined as a decrease in maximal response and/or sensitivity (potency) to insulin (9). Since insulin and the J3-adrenergic agonists have opposing actions on adipose tissue metabolism, we hypothesize that insulin resistance in adipose tissue may be a component of the J3-adrenergic-induced decrease in fat accretion. Beta-adrenergic-induced insulin resistance in adipocytes results, in part, from reduced insulin binding. Insulin receptor number on rodent (1 0,1 1) and swine (12) adipocytes were decreased following short exposures in vitro to ~--adrenergic agonists and other agents which increase the intracellular concentration of ~ P . Little effort has been devoted to confirming whether responses observed in vitro occur in the live animal. In a previous study, this lab was unable to demonstrate insulin resistance in adipocytes from clenbuterol-fed mice, even though resistance was demonstrable in vitro (7). Quantitation of insulin binding kinetics may be a more sensitive measure of ~-adrenergic responses, therefore, one objective of these experiments was to quantify the insulin binding capacity of adipocytes exposed to clenbuterol in vitro and in vivo. Since the efficacy of ractopamine to increase the lean:fat ratio in mice CopynQht© 1~0 by DOMENDO.INC.

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has not been examined, a second objective was to compare the effects of ractopamine and clenbuterol on growth, body composition and insulin binding. MATERIALS AND METHODS A n i m a l s a n d m a n a g e m e n t . Male mice (Swiss albino derived) from the Purdue Animal Sciences colony were housed 4 animals per cage with wood shavings for bedding. Mice from different litters were randomized at weaning (3 weeks) and allotted to cages. Environmental temperature was maintained at 22 C and lights were on from 0800 to 2000. Mice were killed between 0900 and 1000 and were in the early post-feeding state. Mice had ad libitum access to a pelleted commercial rodent diet containing 24% protein and 4% fat (Rodent Blox, Wayne Pet Foods, Chicago IL 60606). Mice weighed 25-35 g for both the in v i t r o studies and the feeding studies. For feeding studies, twelve cages of mice were allotted to one of four treatments (3 replicates) with the ~-adrenergic agonists added to the drinking water. Treatments were: 1) control, 2) 10 mg clenbuterol/liter, 3) 50 mg ractopamine/liter, and 4) 500 mg ractopamine/liter and were offered for 5 days. Doses were selected to give approximate intakes of .05, .25 and 2.5 mg/ day of clenbuterol, ractopamine-50 and ractopamine-500, respectively. Mice were killed by cervical dislocation and weights of the epididymal fat pads, and the skinned hind limb, soleus and gastrocnemius muscles from one leg were taken. Weights of the fat pad and hind limb reflect carcass lipid and lean, respectively (7). Cage means for each variable were used for statistical analysis. Tissue p r o c e s s i n g a n d i n s u l i n b i n d i n g . Adipocytes from epididymal fat pads were prepared by the method of Rodbell (13) as described previously (7) with the exception that media contained .5 mM ascorbic acid and only 1% BSA. Fat pads from 4 mice (1 cage) for each treatment were pooled in the feeding study for adipocyte isolation. C e l l concentration in the final cell suspensions was quantified by a modification of the method of Di Girolamo et al. (14) as described previously (15). Final suspensions contained 4 to 5 X 105 cells/ml. For insulin binding, duplicate .5 ml aliquots of the cell suspension were preincubated at 37 C for 30 min in 17 )< 100 mm polypropylene tubes in a gyratory water bath in an atmosphere of 5% CO2 in oxygen. Media additions of the [3-adrenergic agonists were as indicated in each figure and table. No additions to the media were made for cell incubations from mice given agonists in v i v o . Following preincubation, cells were cooled to 30 C for 5 min and insulin binding was initiated by the addition of porcine 125I-insulin 4 and unlabelled insulin s (0-25 ng/ml). After 1 hr of incubation at 30 C, adipocytes were separated from the media by centrifugation o f . 15 ml aliquots of the cell suspension in triplicate through .1 ml silicone oil (density-----.963) in polyethylene microcentrifuge tubes for 90 sec at 7,300 X g. The cell layer was counted for gamma irradiation 6 and specific binding was calculated by subtracting non-specific binding determined in the presence of excess (. 1 mg/ ml) unlabelled insulin. Insulin binding was expressed as pg insulin bound / 2xlO 5 cells. Data were analyzed using the general linear models procedure of SAS (16). When appropriate, means separation was accomplished by Student-NewmanKuels test.

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Fig. 1. Effect of 10 ~ M ractopamine (PACT) and clenbuterol (CB) on insulin binding to mouse adipocytes (left panel). Adipocytes were incubated for 30 min in the p r e s e n c e of agonist before the addition of 1=51-insulin for the measurement of insulin binding. Scatchard plots (fight panel) w e r e d e r i v e d from the insulin binding data in the left panel. Values are means ± SEM for 3 replicates. Means for ractopamine differed (P<.05) from control at all insulin concentrations. Means for clenbuterol differed (P<.05) from control at 1 and 5 ng insulin/ml and P<.15 at 2, 10 and 25 ng insulin/ml.

RESULTS Incubation of adipocytes for 30 min at 37 C in the presence of 10 .6 M ractopamine or clenbuterol resulted in decreased binding of insulin at all insulin concentrations from 1 to 25 ng/ml (Figure 1). Decreased binding was more pronounced when ractopamine (30%) was present than when clenbuterol (20%) was present. Scatchard plots of the binding curves were characteristically curvilinear (Figure 1). That plots were near parallel suggest that the decrease in insulin binding was due to a decrease in receptor density. The relative potency of ractopamine and clenbuterol to decrease insulin binding was assessed by titrating with the two agonists (Figure 2). Responses to both agonists were similar, therefore a common line was used to describe the relationship. Agonists decreased insulin binding in a dose response, but A

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TABLE 1. EFFECTOF CLENBUTEROLANDRACTOPAMINEON GROWTHANDSELECTEDTISSUEWEIGHTS. Clenbuterol I Ractopamine Variable Control 10 mg/liter 50 500 SEM Initial weight (g) 32.72 32.0 32.1 32.1 .8 Weight gain (g) 1.6 3.3" 1.8 2.4 .5 Fat pad 3 (mg) 342 253" 291 262" 40 Lower hind limb3 (rag) 424 467' 423 416 13 Gastrocnemius 3 (rag) 147 169" 149 143 6 Soleus3 (rag) 9.7 11.6" 10.0 10.5 .7 Gastroc/fat pad .430 .668" .512 .546 .032 IAgonists were added to the drinking water. Mice had ad libitum access for 5 days. zValues are means of 3 cages, 4 mice per cage. 3Weights are for 1 epididymal fat pad or muscle from 1 leg. The lower hind limb was skinned and excised at the distal end of the femur and tibia. 'P<.05 vs. control. the r e s p o n s e a p p e a r e d to have dual c o m p o n e n t s . At c o n c e n t r a t i o n s o f agonists f r o m 10 "13 to 10 .9 M, insulin b i n d i n g was d e c r e a s e d a p p r o x i m a t e l y 10%. At c o n c e n t r a t i o n s g r e a t e r t h a n 10 .9 M, insulin b i n d i n g was f u r t h e r d e c r e a s e d to similar m a x i m a l r e s p o n s e s (30% at 10 .5 M). C o n c e n t r a t i o n s greater than 10 "~ M w e r e not e x a m i n e d . P r o p r a n o l o l p r e v e n t e d the d e c r e a s e in insulin b i n d i n g i n d u c e d b y 10 .7 M r a c t o p a m i n e a n d c l e n b u t e r o l (Figure 2). Administration o f c l e n b u t e r o l in the drinking w a t e r o f m i c e i n c r e a s e d b o d y w e i g h t gain two-fold (Table 1). Gains w e r e p r e d o m i n a n t l y in the lean tissue mass as e v i d e n c e d b y the i n c r e a s e d w e i g h t s o f the l o w e r h i n d l i m b and g a s t r o c n e m i u s and soleus m u s c l e s . E p i d i d y m a l fat pad w e i g h t s w e r e s m a l l e r d e s p i t e the i h c r e a s e d b o d y weight. R a c t o p a m i n e at b o t h dose levels t e n d e d to d e c r e a s e the w e i g h t o f the e p i d i d y m a l fat p a d s ( P < . 0 5 , 500 m g / l i t e r ) w i t h o u t d e c r e a s i n g rate o f gain. N e i t h e r d o s e o f r a c t o p a m i n e affected leg or individual m u s c l e weights. A d i p o c y t e s f r o m m i c e treated w i t h 500 m g / l i t e r r a c t o p a m i n e b o u n d m o r e insulin ( P < . 1) than c o n t r o l m i c e at 1 and 10 ng i n s u l i n / m l (Figure 3). I n c r e a s e d b i n d i n g was a p p a r e n t also at 25 ng i n s u l i n / m l for r a c t o p a m i n e ( 5 0 0 r a g / l i t e r ) a n d for c l e n b u t e r o l at all insulin c o n c e n t r a t i o n s , a l t h o u g h increases w e r e not significant ( P > . 1). R a c t o p a m i n e at 50 r a g / l i t e r did not affect insulin binding. DISCUSSION The ~-adrenergic agonists and insulin have o p p o s i n g actions on a d i p o c y t e m e t a b o l i s m . O n e m e c h a n i s m w h e r e b y the ~-adrenergic agents a p p e a r to dilute the antagonistic actions o f insulin is to directly d i m i n i s h the a d i p o c y t e s res p o n s i v e n e s s to insulin (1 1). N u m e r o u s studies have n o w s h o w n that a v a r i e t y o f ~-adrenergics (11,1"7) or cAMP analogs ( 1 1 , 1 7 ) decrease insulin b i n d i n g and insulin action in r o d e n t a d i p o c y t e s i n v i t r o . Previously w e s h o w e d that the l e a n - p r o m o t i n g agonist c l e n b u t e r o l d e c r e a s e d the sensitivity o f m o u s e a d i p o c y t e s to insulin w h e n m e a s u r i n g fatty acid synthesis i n v i t r o (7). The p r e s e n t results e x t e n d t h e s e p r e v i o u s findings and indicate that c l e n b u t e r o l , as w e l l as r a c t o p a m i n e , decreases the b i n d i n g o f insulin to its m e m b r a n e r e c e p t o r (Figure 1). R e d u c e d insulin b i n d i n g w o u l d b e e x p e c t e d to c o n t r i b u t e to insulin insensitivity o f a d i p o c y t e s ( 9 ) . In the p r e s e n t study, c o n s i s t e n t d e p r e s s i o n s in insulin b i n d i n g w e r e o b s e r v e d w i t h agonist c o n c e n t r a t i o n s g r e a t e r t h a n 10 .9 M ( r a c t o p a m i n e ) or 10 .7 M (clenb u t e r o l ) (Figure 2). We p r e v i o u s l y s h o w e d that c l e n b u t e r o l s t i m u l a t e d lipolysis h a l f - m a x i m a l l y at 5 x l 0 -6 M (7). That similar c o n c e n t r a t i o n s of c l e n b u t e r o l affected lipolysis and insulin b i n d i n g is suggestive that cAMP is i n v o l v e d in

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Fig. 3. Insulin binding to adipocytes from mice g/yen clenbuterol (CB, 10 rag/liter) or ractopamine (RACT, 50 or 500 rag/liter) in the drinking water tor 5 days. Values are means _ SEM for 3 replicates. Means for ractopamine (500 rag/liter) differed ( P < . I ) from control at 1 and 10 ng insulin/ml. Means for clenbuterol d/ffered (P<.2) from control at 1 and 10 n 8 insulin/m1.

mediating both responses. Ractopamine is approximately two orders of magnitude more potent than clenbuterol in stimulating lipolysis in mouse adipocytes (unpublished observations). Differences of that magnitude were not apparent in the present study although ractopamine did appear to have the greater potency. Responses to both agonists were mediated through the [~adrenergic receptor since propranolol prevented the decrease in binding. Both clenbuterol and ractopamine tended to decrease insulin binding (about 10%) at concentrations which w o u l d not be expected to stimulate adenylate cyclase and increase cAMP concentrations (Figure 2, 10 ~3 to 10 .9 M). The possibility that both agonists have membrane associated actions independent of adenylate cyclase cannot be ruled out. We have observed similar responses to ractopamine and clenbuterol in swine adipocytes (12), a tissue which shows high affinity for both agonists yet has a low capacity for adenylate cyclase activation (18). Adipocytes from agonist-treated mice did not show decreased insulin binding, rather, insulin binding tended to be greater in the treatments having the smallest epididymal fat pad weights (clenbuterol and 500 mg ractopamine/liter) (Table 1 and Figure 3). Thus, the importance of decreased insulin binding, as observed in vitro, to the lowered rate of fat accretion in mice fed clenbuterol or ractopamine is questioned and remains unresolved. In agreement with these findings, mice fed clenbuterol did not show reduced sensitivity to insulin or reduced activity of fatty acid synthetase as might be expected ff insulin binding were reduced in chronically fed mice (7). The question arises as to why the discrepancy b e t w e e n the in v i t r o and in v i v o studies? I n vivo, numerous factors interact to influence the net change in insulin receptor concentration, one of the most potent factors being the plasma insulin concentration. Experimental hypoinsulinemia increases insulin receptor concentration (19,20) while hyperinsulinemia decreases insulin receptor concentration (21). Acutely, the ~-adrenergic agonists increase insulin secretion (22), but in animals chronically fed ~-agonists, circulating concentrations of insulin are decreased (23,24). If plasma insulin concentration was reduced in treated mice, it is possible that

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d e c r e a s e d p l a s m a insulin m a s k e d the o p p o s i n g effects of c l e n b u t e r o l and r a c t o p a m i n e on the r e g u l a t i o n o f the insulin r e c e p t o r . The insulin b i n d i n g c a p a c i t y o f a d i p o c y t e s f r o m o b e s e ( o b / o b ) m i c e was i n c r e a s e d after a 2 w k t r e a t m e n t w i t h the ~-agonist BRL 2 6 8 3 0 ( 2 5 ) . H o w these data relate to o u r o w n is u n c e r t a i n since the o b / o b m o u s e is hyperins u l i n e m i c , insulin insensitive and s h o w s r e d u c e d insulin b i n d i n g c o m p a r e d to n o n - o b e s e m i c e . T r e a t m e n t w i t h BRL 2 6 8 3 0 i m p r o v e d in v i v o insulin sensitivity b u t o n l y m a r g i n a l l y c o r r e c t e d the h y p e r i n s u l i n e m i a ( 2 2 ) . T h e r e f o r e , factors o t h e r than d e c r e a s e d insulin c o n c e n t r a t i o n m a y b e i n v o l v e d in the reversal of r e d u c e d insulin b i n d i n g in ~-agonist-treated o b / o b m i c e . Taken w i t h o u r o w n data, it is c l e a r that the effects o f ~-adrenergic agonists on a d i p o c y t e f u n c t i o n are not as straightforward as o b s e r v e d in vitro. In the p r e s e n t studies, r a c t o p a m i n e was far less effective in altering the c o m p o s i t i o n o f g r o w t h than w a s c l e n b u t e r o l . The r e d u c e d effectiveness o f r a c t o p a m i n e was not related to its p o t e n c y to effect the m e t a b o l i s m o f the a d i p o c y t e in v i t r o since r a c t o p a m i n e has e q u a l or greater p o t e n c y t h a n clenb u t e r o l . T h e s e results suggest that ~-agonist actions o n tissues o t h e r t h a n the a d i p o c y t e m a y b e of g r e a t e r i m p o r t a n c e in d e t e r m i n i n g the efficacy of agonists to alter the p a t t e r n o f g r o w t h . W h i l e it is a p p r e c i a t e d that r e d u c e d p o t e n c y in v i v o m a y result f r o m a m y r i a d o f differences in the m e t a b o l i s m o f the t w o agonists, the m o u s e m a y offer an e x p e r i m e n t a l m o d e l to p r o b e w h i c h r e s p o n s e s in v i v o are m o s t i m p o r t a n t in affecting c h a n g e s in b o d y c o m p o s i t i o n . ACKNOWLEDGEMENTS/FOOTNOTES tJournal paper no. 12,137 of the Purdue University Agricultural Experiment Station, West Lafayette, IN. 2The authors thank Boehringer Ingelheim Animal Health, Inc for supplying the clenbuterol and Eli Lilly Corp. for supplying the ractopamine. The secretarial assistance of Melissa Durflinger is gratefully acknowledged. 3Address reprint requests to S. E. Mills, Department of Animal Science, Lilly Hall, Purdue University, West Lafayette, IN 47907. 4Receptor grade insulin, NEX-196 (Mono[t25I] iodotyrAt4, specific activity = 2200 Ci/mmol) NEN Research Products, Boston, MA. 5Calbiochem, Behring Diagnostics, San Diego, CA. 6Packard Instrument Co., Downers Grove, IL. REFERENCES 1. Baker PK, Dalrymple RH, Ingle DL, Ricks CA. Use of a ~-adrenergic agonist to alter muscle and fat deposition in lambs. J Anim Sci 59:1256-1261, 1984. 2. Ricks CA, Dalrymple RH, Baker PK, Ingle DL. Use of a ~-agonist to alter fat and muscle deposition in steers. J Anim Sci 59:1247-1255, 1984. 3. Crenshaw JD, Swantek PM, Marchello MJ, Harrold RL, Zimprich RC, Olson RD. Effects of a phenethanolamine (ractopamine) on swine carcass composition. J Anim Sci 65 (Suppl 1):308, 1987. 4. Hancock JD, Peo ER Jr, Lewis AJ, Parrott JC. Effects of dietary levels of ractopamine (a phenethanolamine) on performance and carcass merit of finishing pigs. J Anim Sci 65 (Suppl 1):309, 1987. 5. Duquette PF, Rickes EL, Olson G, Convey EM. Performance, carcass composition and carcass characteristics of lambs fed the ~-agonist L-644,969. J Anita Sci 65 (Suppl 1):275, 1987. 6. Jones RW, Easter RA, McKeith FK, Dalrymple RH, Maddock HM, Bechtel PJ. Effect of the ~-adrenergic agonist cimaterol (CL 263,780) on the growth and carcass characteristics of finishing swine. J Anim Sci 61:905-913, 1985. 7. Orcutt AL, Cline TR, Mills SE. Influence of the ~2-adrenergic agonist clenbuterol on insulin-stimulated lipogenesis in mouse adipocytes. Domest Anim Endocrinol 6:59-69, 1989.

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8. Smith U, Kuroda M, Simpson IA. Counter-regulation of insulin stimulated glucose transport by catecholamines in the isolated rat adipose cell. J Biol Chem 259:87588763, 1984. 9. Kalm RC. Insulin resistance, insulin insensitivity, and insulin unresponsiveness: A necessary distinction. Metabolism 27:1893-1902, 1978. 10. Sandra A, Marshall SJ. Effect of chronic isoproterenol exposure on insulin binding and insulin-stimulated hexose transport in isolated rat adipocytes. Biochem Biophys Res Comm 148:1093-1097, 1987. 11. PessinJE, Gitomer W, Oka Y, Oppenheimer CC, Czech MP. ~-- adrenergic regulation of insulin and epidermal growth factor receptors in rat adipocytes. J Biol Chem 258:7386-7394, 1983. 12. Liu CY, Mills SE. Decreased insulin binding to porcine adipocytes in vitro by betaadrenergic agonists. J Anita Sci (in press). 13. Rodbell M. Metabolism of isolated fat cells I. Effects of hormones on glucose metabolism and lipolysis. J Biol Chem 239:375-380, 1964. 14. Di Girolamo M, Mendlinger S, Fentig JW. A simple method to determine fat cell size and number in four mammalian species. Am J Physiol 221:850-858, 1971. 15. Mills SE, Orcutt AL. Clenbuterol-induced desensitization in murine adipocytes: Relationship to in vivo effectiveness. Domest Anita Endocrinol 6:51-58, 1989. 16. SAS Institute, Inc. The GLM Procedure. In: SAS User's Guide: Statistics. Statistical Analysis System Institute, Inc, Gary, N.C., 1985. 17. Lonnroth P, Smith U. ~.adrenergic dependent downregulation of insulin binding in rat adipocytes. Biochem Biophys Res Comm 112:972-979, 1983. 18. Liu CY, Mills SE. Determination of the affinity of ractupamine and clenbuterol for the beta-adrenoceptor of the porcine adipocyte. J Anita Sci (in press), 1989. 19. Kasuga M, Akanuma Y, lwamoto Y, Kosaka K. Insulin binding and glucose metabolism in adipocytes of streptozotocin-diabetic rats. Am J Physiol 235:EI75-E282, 1978. 20. Burant CF, Treutelaar MK, Buse MG. Diabetes-induced functional and structural changes in insulin receptors from rat skeletal muscle. J Clin Invest 77:260-270, 1986. 21. Wardzala LJ, Hirshman M, Pofcher E, Horton ED, Mead PM, Cushman SW, Horton ES. Regulation of glucose utilization in adipose cells and muscle after long-term experimental hyperinsulinemia in rats. J Clin Invest 76:460-469, 1985. 22. Smith SA, Levy AL, Sennitt M.A, Simpson DL, Cawthorne MA. Effect of BRL 26830, a novel ~3.adrenoceptor agonist, on glucose tolerance, insulin sensitivity and glucose turnover in Zucker (fa/fa) rats. Biochem Pharmac 34:2425-2429, 1985. 23. Reermann DH, Butler WR, Hogue DE, Fishell VK, Dalrymple RH, Ricks CA, Scanes CG. Cimaterol-induced muscle hypertrophy and altered endocrine status in lambs. J Anita Sci 65:1514-1524, 1987. 24. Emery PW, Rothwell N-J, Stock MJ, Winter PD. Chronic effects of ~z-adrenergic agonists on body composition and protein synthesis in the rat. Biosci Rep 4:8391, 1984. 25. Young P, King L, Cawthorne MA. Increased insulin binding and glucose transport in white adipocytes from C57B1/6 ob/ob mice treated with the thermogenic ~adrenoceptor agonist BRL 26830. Biochem Biophys Res Comm 133:457-461, 1985.