T. A. Peterla2 and C. G. Scanes2 Rutgers - The State University, New Brunswick, NJ 08903 ABSTRACT
The effects of the P-adrenergic agonists isoproterenol, cimaterol, ractopamine and clenbuterol on lipolysis (release of glycerol and free fatty acids) and lipogenesis (incorporation of 14C into fatty acids from [ 14C]glucose) was examined in porcine adipose tissue explants in vitro. Lipolysis was stimulated by isoproterenol, cimaterol or ractopamine but not by clenbuterol. Insulin reduced the lipolytic effects of the P-adrenergic agonists (isoproterenol, cimaterol and ractopamine). Lipogenesis was inhibited by all Padrenergic agonists tested (isoproterenol, cimaterol, ractopamine and clenbuterol). The antilipogenic effect of the -adrenergic agonists was reduced by the presence of insulin in the incubation. Although effects of the different P-adrenergic agonists varied, all had some direct effects that could be expected to reduce adipose accretion. Effects of P-adrenergic agonists in the pig are due in part to direct effects on adipose tissue. (Key Words: Pigs, Lipolysis, Lipogenesis, P-Adrenergic Agonist.)
P
J. h i m . Sci. 1990. 68:1024-1029
corporation into lipid but to be without effect on glucose incorporation into total lipid (Rule Improved feed efficiency, stimulated et al., 1987). Somewhat surprisingly, although growth rate, decreased carcass fat and inclenbuterol reduced carcass fat in pigs in vivo, creased muscle have been reported in pigs with it appeared to be without effect on lipolysis or several P-adrenergic agonists, including cimaother aspects of lipid metabolism in vitro (Rule terol (Jones et al., 1985, Moser et al, 1986), clenbuterol (Van Weerden. 1987), ractopamine et al.. 1987). The present study reexamines (Anderson et al, 1987; Merkel et al., 1987) and the effects of isoproterenol and clenbuterol on L-644,%9 (Wallace et al, 1987). Presumably lipolysis by porcine adipose tissue. Ractopamine has been found to stimulate P-adrenergic agonists exert at least part of their lipolysis and depress lipogenesis by rat adipose partitioning effect in livestock species by directly influencing lipolysis and lipogenesis in tissue in vitro (Hausman et al., 1987). Liu and adipose tissue. Isoproterenol has been found to colleagues (1988) have found that ractopastimulate lipolysis by porcine adipose tissue in mine, in the presence of theophylline, stimuvitro (Mersmann, 1984ab, 1987; Rule et al., lated lipolysis and inhibited insulin-stimulated 1987). The effects of isoproterenol on lipid lipogenesis by porcine adipocytes. The influsynthesis by porcine adipose tissue are less ence of the partitioning agent cimaterol on clear. Isoproterenol has been reported to porcine adipose tissue in vitro has not been reduce palmitate and glycerol-3-phosphate in- determined. The effects of cimaterol and ractopamine on adipose tissue lipolysis in vitro are investigated. In addition, the effects of padrenergic agonists on lipogenesis are evaluat'This is a paper of the Journal Series of the New Jersey ed. These data were reported in part in abstract State AMc. Exp. Sta. (Project 18141)supported by State and Hatch Act funds and a Grant from American Cyanamid. The form (Peterla et al., 1988; Jin et al., 1989). Introduction
authors are grateful to Catherine Ricks for her helpful comments during the course of these studies and to R. L. Jin for assistance in some of the studies. z ~ e p tof . Anim. Sci. Received January 31,1989. Accepted August 17. 1989.
Materials and Methods
Animals and Tissue. Yorkshire x Hampshire crossbred barrows had ad libitum access to
1024
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EFFECT OF PADRENERGIC AGONISTS ON LIPOLYSIS AND LIPOGENESIS BY PORCINE ADIPOSE TISSUE IN VITRO'
p
1025
AGONISTS AND PORCINE FAT METABOLISM
erol-3-phosphate dehydrogenase (8 U; EC 1.1.1.8y were added. Following incubation at room temperature for 10 min, emission again was read. Glycerol release was adjusted for tissue weight and is reported as pmoles glycerol released/@ tissue.h). Release of nonesterified fatty acids was employed as an additional indicator of lipolysis. Nonesterified fatty acids were determined by a micro modification (consisting of a dilution of enzymes by two thirds and a decrease in volume to one fifteenth, utilizing 96-well micro-titer plates) of a spectrophotomemc kit6. Lipogenic rates were determined by the incorporation of 14C into fatty acids from [U''C]glucose. Following incubation, adipose tissue samples were saponified in 10% KOHI 90% methanol at 75°C for 4 h, acidified (to pH 2.5) with HCI, and extracted three times with petroleum ether (7 ml). The petroleum ether was placed into 22-ml scintillation vials and evaporated to dryness. Scintiverse E (15 ml)7 was added and radioactivity was determined in a liquid scintillation counter. Lipogenic rates are presented as pmoles glucose incorporated/ (g tissue.h) based on the incorporation of 14C into fatty acids from [U-14C]glucose and the specific activity of the [~-W]glucose(pCi/ pmol). Chemicals. Cimaterol, clenbuterol and ractopamine were kindly donated, respectively, by American Cyanamid, Boehringer Ingelheim and Eli Lilly. Isoproterenol, ATP, NAD, HEPES, Tris and insulin were obtained from Sigma*. Statistics. Insulin and P-adrenergic agonist effects were determined by a 2 x 2 factorial randomized block plan (blocking by animal) using the General Linear Models procedure of SAS (1985). Differences between control and insulin-alone treatments were determined by t test, using the mean square error determined by the above procedure. Results
P-Adrenergic Agonists. Table 1 summarizes the effects of isoproterenol on lipolysis and lipogenesis. In the absence of insulin, isoprote3Arm0wFraction V, Blue Bell, PA. renol stimulated ( P < .OS> glycerol release to 49.1 mCi/mmol. New England Nuclear. Boston, MA. 'Boehringer Mannheim Biolchemicals. Indianapolis, 5.0- (.l clM> and 5.4-fold (1.0 clM> that of IN. control. Similarly, in the absence of insulin, 6wako. Dallas, TX. isoproterenol increased (P < .05) release of 'Fisher Scientific, Springfield, NJ. free fatty acids (.1 5.4-fold; 1.0 pM, 'Sigma Chemical CO., St. Louis. MO.
w,
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their diet (corn/soybean meal, 18% CP) and were killed at approximately 30 kg by captive bolt gun followed by exsanguination. Subcutaneous adipose tissue was obtained from the dorsal neck region within 5 min of slaughter. The tissue immediately was placed in incubation medium at 37.5"C and transported to the laboratory (I km). Tissue was placed into fresh medium and sliced into explants approximately 1 to 3 mm3 using surgical blades. Incubation. Rates of lipolysis and lipogenesis were determined simultaneously. Adipose tissue explants (five to eight) weighing between 150 and 200 mg were incubated in 2 ml Krebs-Ringer media containing 2% fatty acidfree bovine serum albumin3, 5.0 mM glucose, .1 pCi/ml [U14C]glucose4and 15 mM HEPES buffer. Tissue was incubated under 5 % Coz/ 95% 02,at 37.5'C in a water bath shaking at 60 oscillations per minute. After a 1-h incubation in the presence of various treatments (six replicates/treatment), tissue and media were rapidly cooled by placing them in an acetonehce bath. Tissue and media were immediately separated, frozen and stored at -2O'C prior to assay. Doses of P-adrenergic agonists were chosen based on preliminary experiments (data not shown), and the dose of insulin was similar to that reported previously (Walton, and Etherton, 1986). Each experiment was repeated three or more times. Sample Processing. The rate of lipolysis was estimated by glycerol and free fatty acid release into the media. Glycerol was measured by a fluoromemc method (Wieland, 1983) following deproteinization of the incubation media with an equal volume of 1 N perchloric acid and neutralization with K2CO3. Glycerol determination entailed addition of .4ml media sample to 1.6 ml reaction buffer. The reaction buffer consisted of .125 M Tris, 12.5 mM MgC12, and .5 M hydrazine, pH 9.3, 4 mM ATP and 1.25 mg/ml NAD. Background emission was read (excitation at 340 nm; emission at 450 nni) and 20 pl of a mixture of glycerokinase (.4U; EC 2.7.1.30) and glyc-
1026
PETERLA AND SCANES
TABLE 1. EFFECT OF ISOPROTERENOL (EO)ON P O R C M ADIPOSE TISSUE METABOLISM IN VITRO
0 Insulin LOpf
opt2
IS0
IS0
.388
1.691
2.183
.721
3.881
4.709
1.292
IS 0
1te.m Glycerol released&, pol/(gh) Free fatty acid released& W k h ) Lipogmesis'b, p o l glucose incorporated/(g.h)'
30 nghnl Insulin
. l W IS0
. 1 w IS0
LOW IS0
.385
1.377
1.328
.059
5.351
,775
2.395
3.482
,536
923
4.005
1.994
1.857
SEM
1.25
"Significant ( P e .OS) isoproterenol effect. bsignificant (P e .OS) insdin effect. CBasedon incorporation of 14C into fatty acid from [U-'4C]glucosc.
7.Cfold). Incorporation of [U-''C]glucose into fatty acids was decreased ( P c .OS) by the presence of isoproterenol 72.6% at .1 pM and 80.3% at 1.OpA4. In the absence of insulin, cimaterol at 3 @-f stimulated (P < .05) glycerol release (Table 2) 3.0-fold, as did 30 pM cimaterol (3.2-fold). Cimaterol also stimulated (P c .05) free fatty acid release (2.6-fold at 3 phf and 2.8-fold at 30 pM). Cimaterol reduced (P < .OS) the rate of lipogenesis (73% at 3 pbl and 66.9% at 30 ClM). In contrast to the stimulation of lipolysis observed with isoproterenol and cimaterol, clenbuterol in the absence of insulin did not significantly influence either glycerol or free fatty acid release (Table 3). Clenbuterol depressed (P c .05) the lipogenic rate (64.1% at @-f and 61.9% at 30 ClM). Ractopamine (1 to 30 pM), in the absence of insulin, elevated (P < .05) glycerol release
an average 3.2-fold by porcine adipose tissue explants in vitro (Table 4). Fatty acid synthesis was depressed (P < .05) in the presence of all doses of ractopamine. Fatty acid synthesis was depressed (P c .05) in the presence of all doses of ractopamine. Insulin. Insulin (30 ng/ml) had no significant effect on basal lipolysis as indicated by the absence of a change in glycerol (Tables 1 4 ) or free fatty acid (Tables 1-3) release into the media. Stimulation of glycerol or free fatty acid release by isoproterenol and ractopamine was reduced (P < .05) by the presence of insulin (Tables 1 and 4). Moreover, cimaterolstimulated lipolysis was completely inhibited (P e .05) in the presence of insulin (Table 2). Insulin had no consistent effect on basal fatty acid synthesis (Table 1) except in one experiment in which insulin stimulated lipogenesis (Table 4). Insulin decreased (P < .05)the
TABLE 2. EFFECT OF CIMATEROL (CIM) ON PORCINE ADPOSE TISSUE METABOLISM IN VITRO
0 Insulin Item Glycerol releasedabc, W0Wg.h) Free fatty acid releasedabc, W(gW Lipgenesisah p o l glucose incorporated/(g.h)d
30 n&d Insulin
ow CIM
3 w CIM
3ow
.388
1.004
1.075
.721
2.184
4.709
1.272
ow
3 w CIM
3 o w CIM
SEM
.385
S38
,517
,011
2.393
.775
,863
,826
,101
1.557
4.005
1.972
2.306
1.234
CIM
*Significant ( P c .05) cimaterol effect. bSignificant ( P e .05) insulin effect. 'Significant ( P c .05) cimaterol-insulin interaction. dBased on incopration of 14C into fatty acid from [U-'4C]glucosc.
CIM
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ow
0
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AGONISTS AND PORCINE FAT METABOLISM
TABLE 3. EFFECT OF CLENBUTEROL (CLEN) ON PORCINE ADIPOSE TISSUE METABOLISM IN VITRO ~~
30 ng/rnl Insulin
0 Insulin
OW
CLEN
CLEN
3 W CLEN
3OW CLEN
Glycerol released, pm0Wg.h) Free fatty acid released, M4g.h) Lipogenesisak, pmol glucose incorporated/(gh)d
.388
.241
261
,385
,323
,379
,006
.721
1.038
,970
,775
.7&1
,787
,016
4.709
1.691
1.793
4.005
4.739
4.4%
1.356
F
SEM
‘Data was transformed to log10 for staustical analysis due to high variability. bSignificant (P< .05) clenbuterol effect on loglo transformed data ‘Significant (P< .05) insulin effect on loglo transformed data. dBased on incorporation of 14C into fatty acid from [U-’4C]glucose
magnitude of the inhibition of lipogenesis observed in the presence of isoproterenol (Table 1 ) and cimaterol (Table 2). Furthermore, insulin reduced (P < .05) the depression of lipogenesis seen in the presence of clenbuterol (Table 3) and ractopamine (Table 4). Discussion
P-adrenergic agonists reduce carcass fat in swine. If this were due to a direct effect on adipose tissue, a stimulation of lipolysis might be expected. P-adrenergic receptors have been found on porcine adipose tissue cells (Bocklen et al., 1986). The present studies examined the lipolytic responses to J.3-adrenergic agonists known to affect carcass fat in pigs. Mersmann and colleagues have examined extensively the effects of P-adrenergic agonists on lipolysis in pigs (e.g., Mersmann et al., 1974; Mersmann, 1984a,b 1987). Both isoproterenol and clenbuterol have been found to stimulate lipolysis in
vivo (Mersmann, 1987). However, somewhat surprisingly, whereas isoproterenol stimulated lipolysis in vitro, clenbuterol did not affect lipolysis (Hu et al., 1987). Some p1 and p2 adrenergic agonists ( e g metaphrine, quineren01, Tazolol) have lipolytic activity in vitro with porcine adipose tissue (Mersmann, 1984a; Hu et al.. 1987). In the present studies, isoproterenol, ractopamine and cimaterol stimulated in vitro lipolysis as indicated by an increased release of fatty acids and(or) glycerol (Tables 1, 2 and 4). However, clenbuterol did not influence in vitro lipolysis (Table 3). These observations with clenbuterol and isoproterenol are in full agreement with those reported previously (Hu et al., 1987). Basal and isoproterenol-stimulated rates of lipolysis, as indicated by free fatty acid release, were very similar to those reported recently by Rule et al. (1987). If P-adrenergic agonists exert their partitioning effect by stimulation of lipolysis, then the in vitro lipolytic activities of cimaterol
TABLE 4. EFFECT OF RACTOPAMINE (RAC) ON PORCINE ADIPOSE TISSUE METABOLISM IN VITRO
I tem Glycerol releasedab, m0Vg.h) Lipogenesisab, pmol/glucose incorporated/(gh)‘
0 Insulin 3 w lOW30pM RAC RAC RAC
0p.M
.544
,428
,144
1.667
1.632
Ow
lW
RAC
RAC
,146
,391
,519
1.563
1.663
2.14
RAC
2.63
aSignificant ( P < .OS) ractopamine effect. bSignificant (P < .05) insulin effect ‘Based on incorporation of 14C into fatty acid from [U-14C]glucose.
30 ng/ml Insulin 1 p M 3p.M l O w 3 0 p M RAC RAC RAC RAC .295 2.12
,354 2.08
.376
2.05
,336
2.04
SEM
.003 OR
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3 w CLEN
30w
CLEN
O
Item
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PETERLA AND SCANES
Insulin lowered the analipogenic effects of isoprotenenol and cimaterol (Tables 1 and 2, but it did not completely block the effects of these as it did with clenbuterol (Table 3) and ractopamine (Table 4). Implications
The P-adrenergic agonists, isoproterenol, cimaterol, clenbuterol and ractopamine, have direct effects on porcine adipose tissue, all decreasing lipogenesis. Effects on lipogenesis were lower with insulin present. Effects on lipolysis differed among agonists, with all but clenbuterol increasing lipolysis. Literature Cited
Anderson. D. B., E. L. Veenhuizen. W. P. Waitt, R. E. Paxton and D. H. Mowrey. 1987. Effect of ractopamine on nitrogen retention, growth performance and carcass composition of finisher pigs. J. h i m . Sci. 65(Suppl. 1):130 (Abstr.). Bccklcn. E.,S. Rad, E. Muller and H. Von Faber. 1986. Comparative determination of beta-adrenergic receptors in muscle, h e m and backfat of Pietrain and large while pigs. A n i . Prod. 43:335. Cromwell. G. L., J. D. Kemp. T. S . Stahly and R. H. Dalrymple. 1988. Effects of dietary level and withdrawal time on the efficacy of cimaterol as a growth repaztitioning agent in finishing swine. J. Anim. Sci. 662193. Hausnan. D. B..R. J. Martin,E. L. Veenhuizen and D. B. Anderson. 1987.Effect of phenethanolamine(ractopamine) on in vitro insulin sensitivity and responsiveness of isolated rat adipocytes. Fed. Roc. 46:1178 (Abstr.). Hu, C. Y.,J. Novakofski and H. J. Mersmann. 1987. Honnonal control of porcine adipose tissue fatty acid release and cyclic AMP concentration. J. Anim. Sci. 64:1031. Jin, R.L., T. A. Peterlaand C. G. Scanes. 1989. Ractopamine stimulation of in vitro lipolysis and inhibition of lipogenesis by porcine adipose tissue. FASEB J. 3: A1257 (Abstr.). Jones,R. W.. R. A. Easter, F. K.McKeith. R. H. Dalrymple, H.M. Maddock andP. J. Bechtel. 1985.Effect ofthe padrenergic agonist cimaterol (CL 263.780) on the growth and carcass characteristics of finishing swine. J. Anim. Sci. 61:905. Liu, C. Y., J. L. Boyer and S . E. Mills. 1988. Betaadrenergic agonist inhibition of insulin-stimulated lipogenesis in porcine adpcytes. 1. Anim. Sci. 66(Suppl. 1):249(Abstr.). Merkel, R. A,, R. L. Burkett, R. J. Bumett, P. S. Dickerson, S. E. Johnson, A. L. Schroeder and W. G. Bergen. 1987. The effect of ractopamine on subcutaneous adipose tissue metabolism in pigs. Michigan State Univ. Res. Rep. 487:143. Mersmann,H. J. 1984a. Adrenergic control of lipolysis in swine adipose tissue. Comp. Biochem. Physiol. 77L: 43. Mersmann, H. J. 1984b. Specificity of P-adrenergic control of lipolysis in swine adipose tissue. Comp. Biochern.
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and ractopamine are consistent with the reduction of carcass fat observed in vivo (Jones et al., 1985; Moser et al., 1986 Cromwell et al., 1988). However, the in vivo effects of clenbuterol (Van Weerdon, 1987) cannot be explained by direct stimulation of lipolysis. In the present studies, insulin decreased the lipolysis stimulated by &adrenergic agonists. However, insulin did not affect the basal rate of lipolysis (Table 1). These data are in agreement with those reported previously (Mersmann and Hu, 1987). Whereas the effect of &adrenergic agonists on lipolysis in pig adipose tissue has received considerable attention, less attention has been focused on lipid synthesis. Isoproterenol has been reported to depress incorporation of glycerol-3-phosphate and [I4C]palmitate into total lipid, but no other P-adrenergic agonists have been found to have similar effects (Rule et al., 1987). In the present study, all padrenergic agonists tested (isoproterenol, cimaterol, clenbuterol and ractopamine) inhibited the basal rate of incorporation of I4C into fatty acid from [U14C]glucose. This is the first report of isoproterenol, cimaterol and clenbuterol inhibiting fatty acid synthesis by pig adipose tissue in vitro. The basis for clenbuterol’s inhibiting lipogenesis but not influencing lipolysis is unclear. Perhaps two sub-populations of P-adrenergic receptors are involved. Alternatively. the lipogenic pathway may be more sensitive to P-adrenergic modulation. Insulin (in the absence of P-adrenergic agonists) did not have a consistent effect of lipogenesis in the present studies (Tables 1 and 4). Although some investigators have reported that insulin stimulated lipogenesis (Walton and Etherton, 1986), others have not (O’Hea and Leveille 1968; Mersmann and Hu, 1987). The inconsistence of an insulin effect on basal lipogenesis (Table 1) may reflect different basal lipogenic rates. Presumably the adipose tissue used in the present studies had a high rate of lipogenesis. Subcutaneous adipose tissue from the dorsal neck has greater lipogenic activity than tissue from other regions (O’Hea and Leveille, 1968). Moreover, adipose tissue was obtained from pigs at the age when maximal lipogenic activities have been reported (Mersmann et al., 1973a,b). Lipogenic rates, as measured in these studies by [ 14C]glucose incorporation/(g tissue-h), are similar to those found previously (O’Hea and leveille, 1968; Mersmann et al., 1973a).
fi
AGONISTS AND PORCINE FAT METABOLISM
lipolysis and lipogenesis in porcine adipose tissue in vitro. J. Anim. Sci. 66 (Supple. 1):249 (Abstr.). Rule, D. C., S. B. Smith and H. J . Mersmann. 1987. Effects of adrenergic agonists and insulin on porcine adipose tissue metabolism in vitro. J. Anim. Sci. 65:136. SAS. 1985. SAS User’s Guide: Statistics. SAS Inst., Inc., Cary, NC. Van Weerdon, E. 1. 1987. Effects of clenbuterol on N deposition and carcass composition in castrated mzle pigs. In: J. P. Hanrahan (Ed.) Beta-Agonists and Their Effects on Animal Growth and Carcass quality. pp 152-162. Elsevier Applied Science, London. Wallace, D. H.,H. B. Hedrick, R. L. Seward, C. P. Daurio and E. M. Convey. 1987. Growth and efficiency of feed utilization of swine fed a beta-adrenergic agonist. (L-644, 969). ln: 1. P. Hanrahan (Ed.) Beta-Agonists and Their Effects on Animal Growth and Carcass Quality. pp 143-151. Elsevier Applied Science, London. Walton, P. E. and T. D. Etherton. 1986. Stimulation ol lipogenesis by insulin in swine adipose tissue: Antagonism by porcine growth hormone. J. Anim. Sci. 62:1584. Wieland, 0. 1983. Glycerol UV-method. In: H. U. Bergmeyer J. Bergmeyer and M. Grass1 (Ed.) Method of Enzymatic Analysis (3rd E%.). Vol. 6 pp 504-510. Academic Press, Deerfield Beach, FL.
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Physiol. 77C:39. Mersmann. H. I. 1987. Acute metabolic effects of adrenergic agents in swine. Am. J. Physiol. 252:E85. Mersmann, H.I., L. J. Brown, M.C. Underwood and H. C. Stanton. 1974. Catacholacnine-induced lipolysis in swine. Comp. Biochem. Physiol. 47B:263. Mcrsmann, H.J., J. M.H0uk.G. Phinney. M. C. Undenuood and L. J. Brown. 1973a. Lipogenesis by in vitro liver and adipose tissue preparations from neonatal swine. Am. J. Physiol. 224:1123. Mersmann, H. 1. and C. Y. Hu. 1987. Factors affecting measurements of glucose metabolism and lipolytic rates in porcine adipose tissue slices in vitro. J. Anim. Sci. 64:148. Mersmann,H. J., M. C. Underwood, L. J. Brown and I. M. Hod. 1973b. Adipose tissue composition and lipogenic capacity in developing swine. Am. J. Physiol. 224: 1130. Moser, R. L., R. H. Dalrymple, S. G . Cornelius, J. E. Pettigrew and C. E. Allen. 1986. Effect of c i m a r o l (CL 263,780) as a repartitioning agent in the diet of finishing pigs. J. Anim. Sci. 6221. O’Hea. E.K. and G. A. Leveille. 1968. Lipid metabolism in isolated adipose tissue of the domestic pig (sus domesticus). Comp. Biochem. Physiol. 26: 1081. Peterla, T. A.. C. A. Ricks and C. G. Scanes. 1988. Cimaterol. a b-adrenergic agonist, directly affects
1029
The effects of the P-adrenergic agonists isoproterenol, cimaterol, ractopamine and clenbuterol on lipolysis (release of glycerol and free fatty acids) and lipogenesis (incorporation of 14C into fatty acids from [ 14C]glucose) was examined in porcine adipose tissue explants in vitro. Lipolysis was stimulated by isoproterenol, cimaterol or ractopamine but not by clenbuterol. Insulin reduced the lipolytic effects of the P-adrenergic agonists (isoproterenol, cimaterol and ractopamine). Lipogenesis was inhibited by all Padrenergic agonists tested (isoproterenol, cimaterol, ractopamine and clenbuterol). The antilipogenic effect of the -adrenergic agonists was reduced by the presence of insulin in the incubation. Although effects of the different P-adrenergic agonists varied, all had some direct effects that could be expected to reduce adipose accretion. Effects of P-adrenergic agonists in the pig are due in part to direct effects on adipose tissue. (Key Words: Pigs, Lipolysis, Lipogenesis, P-Adrenergic Agonist.)
P
J. h i m . Sci. 1990. 68:1024-1029
corporation into lipid but to be without effect on glucose incorporation into total lipid (Rule Improved feed efficiency, stimulated et al., 1987). Somewhat surprisingly, although growth rate, decreased carcass fat and inclenbuterol reduced carcass fat in pigs in vivo, creased muscle have been reported in pigs with it appeared to be without effect on lipolysis or several P-adrenergic agonists, including cimaother aspects of lipid metabolism in vitro (Rule terol (Jones et al., 1985, Moser et al, 1986), clenbuterol (Van Weerden. 1987), ractopamine et al.. 1987). The present study reexamines (Anderson et al, 1987; Merkel et al., 1987) and the effects of isoproterenol and clenbuterol on L-644,%9 (Wallace et al, 1987). Presumably lipolysis by porcine adipose tissue. Ractopamine has been found to stimulate P-adrenergic agonists exert at least part of their lipolysis and depress lipogenesis by rat adipose partitioning effect in livestock species by directly influencing lipolysis and lipogenesis in tissue in vitro (Hausman et al., 1987). Liu and adipose tissue. Isoproterenol has been found to colleagues (1988) have found that ractopastimulate lipolysis by porcine adipose tissue in mine, in the presence of theophylline, stimuvitro (Mersmann, 1984ab, 1987; Rule et al., lated lipolysis and inhibited insulin-stimulated 1987). The effects of isoproterenol on lipid lipogenesis by porcine adipocytes. The influsynthesis by porcine adipose tissue are less ence of the partitioning agent cimaterol on clear. Isoproterenol has been reported to porcine adipose tissue in vitro has not been reduce palmitate and glycerol-3-phosphate in- determined. The effects of cimaterol and ractopamine on adipose tissue lipolysis in vitro are investigated. In addition, the effects of padrenergic agonists on lipogenesis are evaluat'This is a paper of the Journal Series of the New Jersey ed. These data were reported in part in abstract State AMc. Exp. Sta. (Project 18141)supported by State and Hatch Act funds and a Grant from American Cyanamid. The form (Peterla et al., 1988; Jin et al., 1989). Introduction
authors are grateful to Catherine Ricks for her helpful comments during the course of these studies and to R. L. Jin for assistance in some of the studies. z ~ e p tof . Anim. Sci. Received January 31,1989. Accepted August 17. 1989.
Materials and Methods
Animals and Tissue. Yorkshire x Hampshire crossbred barrows had ad libitum access to
1024
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EFFECT OF PADRENERGIC AGONISTS ON LIPOLYSIS AND LIPOGENESIS BY PORCINE ADIPOSE TISSUE IN VITRO'
p
1025
AGONISTS AND PORCINE FAT METABOLISM
erol-3-phosphate dehydrogenase (8 U; EC 1.1.1.8y were added. Following incubation at room temperature for 10 min, emission again was read. Glycerol release was adjusted for tissue weight and is reported as pmoles glycerol released/@ tissue.h). Release of nonesterified fatty acids was employed as an additional indicator of lipolysis. Nonesterified fatty acids were determined by a micro modification (consisting of a dilution of enzymes by two thirds and a decrease in volume to one fifteenth, utilizing 96-well micro-titer plates) of a spectrophotomemc kit6. Lipogenic rates were determined by the incorporation of 14C into fatty acids from [U''C]glucose. Following incubation, adipose tissue samples were saponified in 10% KOHI 90% methanol at 75°C for 4 h, acidified (to pH 2.5) with HCI, and extracted three times with petroleum ether (7 ml). The petroleum ether was placed into 22-ml scintillation vials and evaporated to dryness. Scintiverse E (15 ml)7 was added and radioactivity was determined in a liquid scintillation counter. Lipogenic rates are presented as pmoles glucose incorporated/ (g tissue.h) based on the incorporation of 14C into fatty acids from [U-14C]glucose and the specific activity of the [~-W]glucose(pCi/ pmol). Chemicals. Cimaterol, clenbuterol and ractopamine were kindly donated, respectively, by American Cyanamid, Boehringer Ingelheim and Eli Lilly. Isoproterenol, ATP, NAD, HEPES, Tris and insulin were obtained from Sigma*. Statistics. Insulin and P-adrenergic agonist effects were determined by a 2 x 2 factorial randomized block plan (blocking by animal) using the General Linear Models procedure of SAS (1985). Differences between control and insulin-alone treatments were determined by t test, using the mean square error determined by the above procedure. Results
P-Adrenergic Agonists. Table 1 summarizes the effects of isoproterenol on lipolysis and lipogenesis. In the absence of insulin, isoprote3Arm0wFraction V, Blue Bell, PA. renol stimulated ( P < .OS> glycerol release to 49.1 mCi/mmol. New England Nuclear. Boston, MA. 'Boehringer Mannheim Biolchemicals. Indianapolis, 5.0- (.l clM> and 5.4-fold (1.0 clM> that of IN. control. Similarly, in the absence of insulin, 6wako. Dallas, TX. isoproterenol increased (P < .05) release of 'Fisher Scientific, Springfield, NJ. free fatty acids (.1 5.4-fold; 1.0 pM, 'Sigma Chemical CO., St. Louis. MO.
w,
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their diet (corn/soybean meal, 18% CP) and were killed at approximately 30 kg by captive bolt gun followed by exsanguination. Subcutaneous adipose tissue was obtained from the dorsal neck region within 5 min of slaughter. The tissue immediately was placed in incubation medium at 37.5"C and transported to the laboratory (I km). Tissue was placed into fresh medium and sliced into explants approximately 1 to 3 mm3 using surgical blades. Incubation. Rates of lipolysis and lipogenesis were determined simultaneously. Adipose tissue explants (five to eight) weighing between 150 and 200 mg were incubated in 2 ml Krebs-Ringer media containing 2% fatty acidfree bovine serum albumin3, 5.0 mM glucose, .1 pCi/ml [U14C]glucose4and 15 mM HEPES buffer. Tissue was incubated under 5 % Coz/ 95% 02,at 37.5'C in a water bath shaking at 60 oscillations per minute. After a 1-h incubation in the presence of various treatments (six replicates/treatment), tissue and media were rapidly cooled by placing them in an acetonehce bath. Tissue and media were immediately separated, frozen and stored at -2O'C prior to assay. Doses of P-adrenergic agonists were chosen based on preliminary experiments (data not shown), and the dose of insulin was similar to that reported previously (Walton, and Etherton, 1986). Each experiment was repeated three or more times. Sample Processing. The rate of lipolysis was estimated by glycerol and free fatty acid release into the media. Glycerol was measured by a fluoromemc method (Wieland, 1983) following deproteinization of the incubation media with an equal volume of 1 N perchloric acid and neutralization with K2CO3. Glycerol determination entailed addition of .4ml media sample to 1.6 ml reaction buffer. The reaction buffer consisted of .125 M Tris, 12.5 mM MgC12, and .5 M hydrazine, pH 9.3, 4 mM ATP and 1.25 mg/ml NAD. Background emission was read (excitation at 340 nm; emission at 450 nni) and 20 pl of a mixture of glycerokinase (.4U; EC 2.7.1.30) and glyc-
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PETERLA AND SCANES
TABLE 1. EFFECT OF ISOPROTERENOL (EO)ON P O R C M ADIPOSE TISSUE METABOLISM IN VITRO
0 Insulin LOpf
opt2
IS0
IS0
.388
1.691
2.183
.721
3.881
4.709
1.292
IS 0
1te.m Glycerol released&, pol/(gh) Free fatty acid released& W k h ) Lipogmesis'b, p o l glucose incorporated/(g.h)'
30 nghnl Insulin
. l W IS0
. 1 w IS0
LOW IS0
.385
1.377
1.328
.059
5.351
,775
2.395
3.482
,536
923
4.005
1.994
1.857
SEM
1.25
"Significant ( P e .OS) isoproterenol effect. bsignificant (P e .OS) insdin effect. CBasedon incorporation of 14C into fatty acid from [U-'4C]glucosc.
7.Cfold). Incorporation of [U-''C]glucose into fatty acids was decreased ( P c .OS) by the presence of isoproterenol 72.6% at .1 pM and 80.3% at 1.OpA4. In the absence of insulin, cimaterol at 3 @-f stimulated (P < .05) glycerol release (Table 2) 3.0-fold, as did 30 pM cimaterol (3.2-fold). Cimaterol also stimulated (P c .05) free fatty acid release (2.6-fold at 3 phf and 2.8-fold at 30 pM). Cimaterol reduced (P < .OS) the rate of lipogenesis (73% at 3 pbl and 66.9% at 30 ClM). In contrast to the stimulation of lipolysis observed with isoproterenol and cimaterol, clenbuterol in the absence of insulin did not significantly influence either glycerol or free fatty acid release (Table 3). Clenbuterol depressed (P c .05) the lipogenic rate (64.1% at @-f and 61.9% at 30 ClM). Ractopamine (1 to 30 pM), in the absence of insulin, elevated (P < .05) glycerol release
an average 3.2-fold by porcine adipose tissue explants in vitro (Table 4). Fatty acid synthesis was depressed (P < .05) in the presence of all doses of ractopamine. Fatty acid synthesis was depressed (P c .05) in the presence of all doses of ractopamine. Insulin. Insulin (30 ng/ml) had no significant effect on basal lipolysis as indicated by the absence of a change in glycerol (Tables 1 4 ) or free fatty acid (Tables 1-3) release into the media. Stimulation of glycerol or free fatty acid release by isoproterenol and ractopamine was reduced (P < .05) by the presence of insulin (Tables 1 and 4). Moreover, cimaterolstimulated lipolysis was completely inhibited (P e .05) in the presence of insulin (Table 2). Insulin had no consistent effect on basal fatty acid synthesis (Table 1) except in one experiment in which insulin stimulated lipogenesis (Table 4). Insulin decreased (P < .05)the
TABLE 2. EFFECT OF CIMATEROL (CIM) ON PORCINE ADPOSE TISSUE METABOLISM IN VITRO
0 Insulin Item Glycerol releasedabc, W0Wg.h) Free fatty acid releasedabc, W(gW Lipgenesisah p o l glucose incorporated/(g.h)d
30 n&d Insulin
ow CIM
3 w CIM
3ow
.388
1.004
1.075
.721
2.184
4.709
1.272
ow
3 w CIM
3 o w CIM
SEM
.385
S38
,517
,011
2.393
.775
,863
,826
,101
1.557
4.005
1.972
2.306
1.234
CIM
*Significant ( P c .05) cimaterol effect. bSignificant ( P e .05) insulin effect. 'Significant ( P c .05) cimaterol-insulin interaction. dBased on incopration of 14C into fatty acid from [U-'4C]glucosc.
CIM
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ow
0
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AGONISTS AND PORCINE FAT METABOLISM
TABLE 3. EFFECT OF CLENBUTEROL (CLEN) ON PORCINE ADIPOSE TISSUE METABOLISM IN VITRO ~~
30 ng/rnl Insulin
0 Insulin
OW
CLEN
CLEN
3 W CLEN
3OW CLEN
Glycerol released, pm0Wg.h) Free fatty acid released, M4g.h) Lipogenesisak, pmol glucose incorporated/(gh)d
.388
.241
261
,385
,323
,379
,006
.721
1.038
,970
,775
.7&1
,787
,016
4.709
1.691
1.793
4.005
4.739
4.4%
1.356
F
SEM
‘Data was transformed to log10 for staustical analysis due to high variability. bSignificant (P< .05) clenbuterol effect on loglo transformed data ‘Significant (P< .05) insulin effect on loglo transformed data. dBased on incorporation of 14C into fatty acid from [U-’4C]glucose
magnitude of the inhibition of lipogenesis observed in the presence of isoproterenol (Table 1 ) and cimaterol (Table 2). Furthermore, insulin reduced (P < .05) the depression of lipogenesis seen in the presence of clenbuterol (Table 3) and ractopamine (Table 4). Discussion
P-adrenergic agonists reduce carcass fat in swine. If this were due to a direct effect on adipose tissue, a stimulation of lipolysis might be expected. P-adrenergic receptors have been found on porcine adipose tissue cells (Bocklen et al., 1986). The present studies examined the lipolytic responses to J.3-adrenergic agonists known to affect carcass fat in pigs. Mersmann and colleagues have examined extensively the effects of P-adrenergic agonists on lipolysis in pigs (e.g., Mersmann et al., 1974; Mersmann, 1984a,b 1987). Both isoproterenol and clenbuterol have been found to stimulate lipolysis in
vivo (Mersmann, 1987). However, somewhat surprisingly, whereas isoproterenol stimulated lipolysis in vitro, clenbuterol did not affect lipolysis (Hu et al., 1987). Some p1 and p2 adrenergic agonists ( e g metaphrine, quineren01, Tazolol) have lipolytic activity in vitro with porcine adipose tissue (Mersmann, 1984a; Hu et al.. 1987). In the present studies, isoproterenol, ractopamine and cimaterol stimulated in vitro lipolysis as indicated by an increased release of fatty acids and(or) glycerol (Tables 1, 2 and 4). However, clenbuterol did not influence in vitro lipolysis (Table 3). These observations with clenbuterol and isoproterenol are in full agreement with those reported previously (Hu et al., 1987). Basal and isoproterenol-stimulated rates of lipolysis, as indicated by free fatty acid release, were very similar to those reported recently by Rule et al. (1987). If P-adrenergic agonists exert their partitioning effect by stimulation of lipolysis, then the in vitro lipolytic activities of cimaterol
TABLE 4. EFFECT OF RACTOPAMINE (RAC) ON PORCINE ADIPOSE TISSUE METABOLISM IN VITRO
I tem Glycerol releasedab, m0Vg.h) Lipogenesisab, pmol/glucose incorporated/(gh)‘
0 Insulin 3 w lOW30pM RAC RAC RAC
0p.M
.544
,428
,144
1.667
1.632
Ow
lW
RAC
RAC
,146
,391
,519
1.563
1.663
2.14
RAC
2.63
aSignificant ( P < .OS) ractopamine effect. bSignificant (P < .05) insulin effect ‘Based on incorporation of 14C into fatty acid from [U-14C]glucose.
30 ng/ml Insulin 1 p M 3p.M l O w 3 0 p M RAC RAC RAC RAC .295 2.12
,354 2.08
.376
2.05
,336
2.04
SEM
.003 OR
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3 w CLEN
30w
CLEN
O
Item
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PETERLA AND SCANES
Insulin lowered the analipogenic effects of isoprotenenol and cimaterol (Tables 1 and 2, but it did not completely block the effects of these as it did with clenbuterol (Table 3) and ractopamine (Table 4). Implications
The P-adrenergic agonists, isoproterenol, cimaterol, clenbuterol and ractopamine, have direct effects on porcine adipose tissue, all decreasing lipogenesis. Effects on lipogenesis were lower with insulin present. Effects on lipolysis differed among agonists, with all but clenbuterol increasing lipolysis. Literature Cited
Anderson. D. B., E. L. Veenhuizen. W. P. Waitt, R. E. Paxton and D. H. Mowrey. 1987. Effect of ractopamine on nitrogen retention, growth performance and carcass composition of finisher pigs. J. h i m . Sci. 65(Suppl. 1):130 (Abstr.). Bccklcn. E.,S. Rad, E. Muller and H. Von Faber. 1986. Comparative determination of beta-adrenergic receptors in muscle, h e m and backfat of Pietrain and large while pigs. A n i . Prod. 43:335. Cromwell. G. L., J. D. Kemp. T. S . Stahly and R. H. Dalrymple. 1988. Effects of dietary level and withdrawal time on the efficacy of cimaterol as a growth repaztitioning agent in finishing swine. J. Anim. Sci. 662193. Hausnan. D. B..R. J. Martin,E. L. Veenhuizen and D. B. Anderson. 1987.Effect of phenethanolamine(ractopamine) on in vitro insulin sensitivity and responsiveness of isolated rat adipocytes. Fed. Roc. 46:1178 (Abstr.). Hu, C. Y.,J. Novakofski and H. J. Mersmann. 1987. Honnonal control of porcine adipose tissue fatty acid release and cyclic AMP concentration. J. Anim. Sci. 64:1031. Jin, R.L., T. A. Peterlaand C. G. Scanes. 1989. Ractopamine stimulation of in vitro lipolysis and inhibition of lipogenesis by porcine adipose tissue. FASEB J. 3: A1257 (Abstr.). Jones,R. W.. R. A. Easter, F. K.McKeith. R. H. Dalrymple, H.M. Maddock andP. J. Bechtel. 1985.Effect ofthe padrenergic agonist cimaterol (CL 263.780) on the growth and carcass characteristics of finishing swine. J. Anim. Sci. 61:905. Liu, C. Y., J. L. Boyer and S . E. Mills. 1988. Betaadrenergic agonist inhibition of insulin-stimulated lipogenesis in porcine adpcytes. 1. Anim. Sci. 66(Suppl. 1):249(Abstr.). Merkel, R. A,, R. L. Burkett, R. J. Bumett, P. S. Dickerson, S. E. Johnson, A. L. Schroeder and W. G. Bergen. 1987. The effect of ractopamine on subcutaneous adipose tissue metabolism in pigs. Michigan State Univ. Res. Rep. 487:143. Mersmann,H. J. 1984a. Adrenergic control of lipolysis in swine adipose tissue. Comp. Biochem. Physiol. 77L: 43. Mersmann, H. J. 1984b. Specificity of P-adrenergic control of lipolysis in swine adipose tissue. Comp. Biochern.
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and ractopamine are consistent with the reduction of carcass fat observed in vivo (Jones et al., 1985; Moser et al., 1986 Cromwell et al., 1988). However, the in vivo effects of clenbuterol (Van Weerdon, 1987) cannot be explained by direct stimulation of lipolysis. In the present studies, insulin decreased the lipolysis stimulated by &adrenergic agonists. However, insulin did not affect the basal rate of lipolysis (Table 1). These data are in agreement with those reported previously (Mersmann and Hu, 1987). Whereas the effect of &adrenergic agonists on lipolysis in pig adipose tissue has received considerable attention, less attention has been focused on lipid synthesis. Isoproterenol has been reported to depress incorporation of glycerol-3-phosphate and [I4C]palmitate into total lipid, but no other P-adrenergic agonists have been found to have similar effects (Rule et al., 1987). In the present study, all padrenergic agonists tested (isoproterenol, cimaterol, clenbuterol and ractopamine) inhibited the basal rate of incorporation of I4C into fatty acid from [U14C]glucose. This is the first report of isoproterenol, cimaterol and clenbuterol inhibiting fatty acid synthesis by pig adipose tissue in vitro. The basis for clenbuterol’s inhibiting lipogenesis but not influencing lipolysis is unclear. Perhaps two sub-populations of P-adrenergic receptors are involved. Alternatively. the lipogenic pathway may be more sensitive to P-adrenergic modulation. Insulin (in the absence of P-adrenergic agonists) did not have a consistent effect of lipogenesis in the present studies (Tables 1 and 4). Although some investigators have reported that insulin stimulated lipogenesis (Walton and Etherton, 1986), others have not (O’Hea and Leveille 1968; Mersmann and Hu, 1987). The inconsistence of an insulin effect on basal lipogenesis (Table 1) may reflect different basal lipogenic rates. Presumably the adipose tissue used in the present studies had a high rate of lipogenesis. Subcutaneous adipose tissue from the dorsal neck has greater lipogenic activity than tissue from other regions (O’Hea and Leveille, 1968). Moreover, adipose tissue was obtained from pigs at the age when maximal lipogenic activities have been reported (Mersmann et al., 1973a,b). Lipogenic rates, as measured in these studies by [ 14C]glucose incorporation/(g tissue-h), are similar to those found previously (O’Hea and leveille, 1968; Mersmann et al., 1973a).
fi
AGONISTS AND PORCINE FAT METABOLISM
lipolysis and lipogenesis in porcine adipose tissue in vitro. J. Anim. Sci. 66 (Supple. 1):249 (Abstr.). Rule, D. C., S. B. Smith and H. J . Mersmann. 1987. Effects of adrenergic agonists and insulin on porcine adipose tissue metabolism in vitro. J. Anim. Sci. 65:136. SAS. 1985. SAS User’s Guide: Statistics. SAS Inst., Inc., Cary, NC. Van Weerdon, E. 1. 1987. Effects of clenbuterol on N deposition and carcass composition in castrated mzle pigs. In: J. P. Hanrahan (Ed.) Beta-Agonists and Their Effects on Animal Growth and Carcass quality. pp 152-162. Elsevier Applied Science, London. Wallace, D. H.,H. B. Hedrick, R. L. Seward, C. P. Daurio and E. M. Convey. 1987. Growth and efficiency of feed utilization of swine fed a beta-adrenergic agonist. (L-644, 969). ln: 1. P. Hanrahan (Ed.) Beta-Agonists and Their Effects on Animal Growth and Carcass Quality. pp 143-151. Elsevier Applied Science, London. Walton, P. E. and T. D. Etherton. 1986. Stimulation ol lipogenesis by insulin in swine adipose tissue: Antagonism by porcine growth hormone. J. Anim. Sci. 62:1584. Wieland, 0. 1983. Glycerol UV-method. In: H. U. Bergmeyer J. Bergmeyer and M. Grass1 (Ed.) Method of Enzymatic Analysis (3rd E%.). Vol. 6 pp 504-510. Academic Press, Deerfield Beach, FL.
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Physiol. 77C:39. Mersmann. H. I. 1987. Acute metabolic effects of adrenergic agents in swine. Am. J. Physiol. 252:E85. Mersmann, H.I., L. J. Brown, M.C. Underwood and H. C. Stanton. 1974. Catacholacnine-induced lipolysis in swine. Comp. Biochem. Physiol. 47B:263. Mcrsmann, H.J., J. M.H0uk.G. Phinney. M. C. Undenuood and L. J. Brown. 1973a. Lipogenesis by in vitro liver and adipose tissue preparations from neonatal swine. Am. J. Physiol. 224:1123. Mersmann, H. 1. and C. Y. Hu. 1987. Factors affecting measurements of glucose metabolism and lipolytic rates in porcine adipose tissue slices in vitro. J. Anim. Sci. 64:148. Mersmann,H. J., M. C. Underwood, L. J. Brown and I. M. Hod. 1973b. Adipose tissue composition and lipogenic capacity in developing swine. Am. J. Physiol. 224: 1130. Moser, R. L., R. H. Dalrymple, S. G . Cornelius, J. E. Pettigrew and C. E. Allen. 1986. Effect of c i m a r o l (CL 263,780) as a repartitioning agent in the diet of finishing pigs. J. Anim. Sci. 6221. O’Hea. E.K. and G. A. Leveille. 1968. Lipid metabolism in isolated adipose tissue of the domestic pig (sus domesticus). Comp. Biochem. Physiol. 26: 1081. Peterla, T. A.. C. A. Ricks and C. G. Scanes. 1988. Cimaterol. a b-adrenergic agonist, directly affects
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