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Inhibition of porcine adipose tissue lipogenesis by β-adrenergic agonists
Vol. 94C,
Camp. Biochem. Physiol.
No. 2, pp. 619-623,
1989
Pergamon Press plc
Printed in Great Britain
INHIBITION LIPOGENESIS
OF PORCINE ADIPOSE TISSUE BY p-ADRENERGIC AGONISTS
HARRY J. MERSMANN US Department of Agriculture, Clay Center, NE 68933 USA* Telephone (7 13) 7987000 (Received 28
July
1989)
Abstract-l. Beta-adrenergic agonists were not effective inhibitors of lipogenesis in porcine adipose tissue slices in vitro; addition of theophylline permitted the inhibition. 2. Inhibition was increased to a greater extent by isoproterenol than epinephrine and was decreased by propranolol, therefore presumably via D-adrenergic receptors. 3. Caffeine, isobutylmethylxanthine and theophylline all permitted inhibition of lipogenesis by /I-adrenergic agonists. 4. It is not clear whether the mechanism for this permissive action is via antagonism of the adenosine receptor, inhibition of CAMP phosphodiesterase or a combination of both. 5. Adenosine deaminase was weakly permissive, presumably through destruction of adenosine. Inhibition of lipogenesis was observed with glucose or acetate as lipogenic substrate and in the presence
or absence of albumin.
INTRODUCTION
adipose tissue lipid synthesis (lipogenesis) is inhibited (Saggerson, 1985) and lipid degradation (lipolysis) is stimulated (Vernon and Clegg, 1985) in vitro by B-adrenergic agonists. Initial studies by this investigator (Mersmann et al., 1974) established that p-adrenergic agonists increase rates of lipolysis in porcine adipose tissue in vitro. Subsequently, it was shown that this tissue responds to a select number of agonists only; it was concluded that the receptor on this tissue exhibits stringent specificity for control of lipolysis and tissue CAMP concentration (Mersmann, 1984a,b; Hu et al., 1987). The specificity can be demonstrated in vivo also (Mersmann, 1987a). Evidence was presented to indicate /3-adrenergic agonists alone, including isoproterenol, do not inhibit lipogenesis in porcine adipose tissue in vitro (Rule et al.. 1987). More direct assessment of aspects of triacylglycerol biosynthesis indicated isoproterenol inhibits but only under specific incubation conditions (Rule et al., 1987). Recently Mills and co-workers (Liu et al., 1989; Liu and Mills, 1989) have confirmed that epinephrine alone is a poor agonist for inhibition of lipogenesis when incubated with isolated porcine adipocytes in vitro. However, if the incubation medium contains theophylline or adenosine deaminase, epinephrine effectively inhibits lipogenesis. The purpose of the studies indicated herein was to determine if observations similar to those of Mammalian
*Agricultural Research Service, Roman L. Hruska, US Meat Animal Research Center. P.O. Box 166. Clav Center, NE, USA. Mention of trade names, proprietary products or specific equipment 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.
Mills and co-workers could be demonstrated in tissues lices (the preparation used in this laboratory) or whether /I-adrenergic agonist inhibition of lipogenesis was observable only after exposure to the proteolytic enzymes used for adipocyte isolation. METHODS Crossbred (l/4 Yorkshire, l/4 Large White, I/4 Chester White, l/4 Landrace) castrated male pigs (Sus domesticus), were used at weights between 20 and 36 kg. Pigs were about 9-15 wk of age and were fed a corn-soybean meal diet containing 18% calculated protein ad libitum,from weaning at 4wk. Adipose tissue biopsies were obtained from the dorsal neck region of pigs anesthetized with pentothal Na, as indicated previously (Mersmann, 1983). Tissue was transported to the laboratory in 0.9% NaCl, 5.6 mM glucose, 25 mM Hepes at pH 7.4 and 35°C. Tissue from an individual animal was used to prepare a pool of 0.4 mm slices with a tissue slicer. Slices from the pool were used to execute an individual replicate of an experiment. Lipogenesis was assessed by measuring incorporation of i4C-glucose into a total lipid extract. The incubation medium contained Krebs-Ringer bicarbonate buffer with 50% of the indicated calcium concentration, 5 mM glucose, 0.5 FCi radioactive glucose, 0.57 mM ascorbate, 1munit insulin/ml and 100 mg tissue slices, unless indicated otherwise. Incubations were in triplicate at 37°C for 120 min, with reciprocal shaking at 90 strokes/min. Reactions were stopped by injection of 0.25 ml 1 N H,SO,; lipids were extracted with chloroform: methanol (2 : 1, v/v); radioactivity in the extract was quantified by liquid scintillation spectrometry. Details of these procedures were reported previously (Mersmann and Hu, 1987). In one experiment, 0.5pCi i4C-acetate was used as substrate. Additions to incubation flasks and modifications to the standard incubation conditions are detailed for each experiment. Isoproterenol bitartrate (12760), theophylline (T’1633), deaminase (A6648), adenosine (A925 I), adenosine (-)epinephrine bitartrate (E4375), 3-isobutyl-l-methylxanthine (15879), caffeine (CO750), m-propranolol HCl (P0884) were purchased from Sigma Chemical Co., St Louis, MO 63178, USA. Bovine serum albumin, Bovuminar
619
HARRYJ. MERSMANN
620
reagent CRG-7 (2295-00) was purchased from Armour Pharmaceutical Co., Kankakee, IL 60901, USA. Crystalline porcine insulin (SI-144-2A; 24.7 units/mg) was generously provided by Lilly Research Laboratory, Greenfield, IN 46140, USA. D-[‘4c(u)]ghCOSe (NEC-042X) and sodium [2-V] acetate (NEC085H) were purchased from NEN Research Products, DuPont Company, Wilmington, DE, 19898, USA.
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RESULTS
The first attempt to replicate the observations of Mills and co-workers involved incubation of tissue slices with epinephrine or epinephrine plus 5 x 10m4M theophylline. There was a tendency for glucose incorporation into both CO, and lipid to increase slightly with increasing epinephrine concentration, whereas in the presence of theophylline there was a tendency for incorporation to decrease with increased epinephrine concentration (data not indicated). This result qualitatively reproduced the observations of Mills and co-workers (Liu et al., 1989), in that lipogenesis was inhibited, albeit to a small extent only, by a /?-adrenergic agonist, epinephrine; theophylline was a necessary medium component for this inhibition. It reproduced the observations of Rule et al. (1987) also, in that there was no inhibition and possibly slight stimulation when theophylline was not in the incubation medium. Epinephrine, an a- plus fi-adrenergic agonist, was compared with isoproterenol, a pure /I-adrenergic agonist; both agonists were studied in the presence of 5 x 10e4 M theophylline (Fig. 1). Epinephrine and isoproterenol each tended to inhibit lipogenesis, with isoproterenol being more potent than epinephrine. The inhibition was only about 30% at the highest concentrations of either agonist. All subsequent experiments used isoproterenol to eliminate the possibility of a-adrenergic effects when utilizing the mixed agonist, epinephrine. Theophylline (5 x 10m4M) alone, i.e. no isoproterenol, did not inhibit insulin-stimulated lipogenesis; glucose incorporation into lipids was 5.94 f 0.18 (3 experiments) in the control flasks and 5.64 f 0.90 in the presence of 5 x 10e4 M theophylline. Liu et al.
0
0.01
0.1
0.6
Theophylline,
1
6
ImMl
Fig. 2. Theophylline saturation. Incubation conditions and data presentation were the same as for Fig. 1 except isoproterenol was at 10m6M and theophylline concentration was varied as indicated. There were two independent replications, each with tissue from a different pig; pigs weighed 19.3 and 22.7 kg.
(1989) indicated that, in the presence of insulin, theophylline alone did not inhibit lipogenesis. Because the magnitude of inhibition of tissue slice lipogenesis by isoproterenol (or epinephrine) was small in the presence of 5 x 10m4M theophylline, the concentration of theophylline was titrated at a fixed concentration ( lO-6 M) of isoproterenol (Fig. 2). Lipogenesis was strongly inhibited by isoproterenol but only at theophylline concentrations >5 x low4 M. These concentrations were greater than are necessary to demonstrate similar effects in isolated porcine cells (Liu et al., 1989). Propranolol, a fi-adrenergic antagonist, reversed the inhibition of lipogenesis by isoproterenol in the presence of theophylline (Fig. 3). Because isoproterenol was more potent than epinephrine and isoproterenol-stimulated inhibition was reversed by propranolol, a pure j?-adrenergic agonist or antagonist, respectively, the inhibition of lipogenesis was almost certainly through the fi-adrenergic receptor. At a medium concentration of lo-‘M, theophylline (T) and the related compound, caffeine (Caf), both permitted the inhibition of lipogenesis by isoproterenol to the same extent (Fig. 4) whereas
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Fig. 1. Inhibition of lipogenesis by epinephrine and isoproterenol. Tissue slices were incubated with 5 mM glucose, 0.57 mM ascorbate, 5 x 1O-4 M theophylline, 1 m unit insulin/ml and epinephrine (hatched bars) or isoproterenol (solid bars) concentrations between lo-* and 10p4M, as indicated. Lipogenesis is expressed as pmol [UJ4C]glucose incorporated into total lipid per 120 min/g tissue. Data are mean with SD indicated as error bars. There were three independent replications, each with tissue from a different pig; pigs weighed 21.8 f 0.5 kg.
-8
-7
Propranolol,
-6
-5
log
-4
[Ml
Fig. 3. Reversal of /I-adrenergic stimulated inhibition of lipogenesis by a fi-adrenergic antagonist. Incubation conditions and data presentation were the same as for Fig. 1, except there was 10w6M isoproterenol and lo-)M theophylline were present in all flasks; propranolol was varied as indicated. There were two independent replications each with tissue from a different pig; pigs weighed 28.7 and 36.0 kg. The uninhibited lipogenic rate [indicated as mean (SD)] was 7.8 (2.3) prnol [II-“‘Clglucose incorporated into total lipid per 120 min/g tissue (10m6M isoproterenol and no theophylline in the Rasks).
/I-Adrenergic inhibition of lipogenesis
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Adenosina Deaminase,
Fig. 4. Miscellaneous effects on isoproterenol inhibition of lipogenesis. Incubation conditions generally were the same as for Fig. 1. Isoproterenol was at lO-‘jM in all flasks including the controls (C); theophylline (T), methylisobutyl xanthine (IBX) and caffeine (Caf) were at lo-)M [theophylline was present only where indicated (T)]; adenosine deaminase (AdD) was at 1 unit/ml; adenosine (Ad) was at IO-‘M; bovine serum albumin (Al) was at 30mg/ml or 3%. Data are indicated as percent of control (C) calculated on an individual animal basis because of divergent control values (8.46 and 4.34 pmol [U-r4C]glucose incorporated into total lipid per 120 min/g tissue); mean with SD indicated as error bars. There were two independent replications each with tissue from a different pig; pigs weighed 29.3 and 32.1 kg.
isobutylmethylxanthine
100
adeno-
sine receptor antagonist and inhibitor of CAMP phosphodiesterase than theophylline or caffeine (Choi et al., 1988), permitted greater inhibition of lipogenesis by isoproterenol. Albumin is used in media to assess lipogenesis by some investigators (see Mersmann and Hu, 1987, for references) and is a necessary medium component in studies with isolated adipocytes to maintain cell integrity. In the presence of albumin, lipogenesis might be indirectly inhibited by isoproterenol, because isoproterenol-stimulated lipolysis would produce free-fatty acids that could cause feedback inhibition of lipogenesis. This was not the case (Fig. 4Al), as was observed previously (Rule et al., 1987Table 1). Isoproterenol inhibited lipogenesis in the presence of albumin, only when theophylline was present (Fig. 4-Al+ T). Addition of lo-’ M adenosine (Ad) had little effect on the isoproterenol response in the absence of theophylline; addition of 1 unit of adenosine deaminase/ ml (AdD) to the isoproterenol-containing medium (no theophylline), tended to permit the inhibition (Fig. 4). Titration of adenosine deaminase concentration, indicated the permission of isoproterenol inhibition of lipogenesis was adenosine deaminase concentration dependent (Fig. 5); however, the maximum inhibition was only about 30% even at extremely high concentrations of the enzyme. Initial studies suggested, that not only was lipogenesis inhibited by epinephrine (in the presence of theophylline), but incorporation of glucose carbon into CO, was inhibited, also. Experiments in this manuscript used 5 mM glucose to mimic the conditions of Mills and co-workers; theophylline permitted isoproterenol-stimulated inhibition of lipogenesis with both 5 (g) or 20 (G) mM glucose as lipogenic substrate (Fig. 6), thus, the inhibition observed by Mills, but not previously by this laboratory (routine use of 20mM glucose), was not the
1000
mu/ml
Fig. 5. Saturation of adenosine deaminase. Incubation conditions were the same as for Fig. 1 except isoproterenol was at 10m6M and there was no theophylline present. Adenosine deaminase was varied as indicated. These were the same two pigs as in Fig. 3.
result of glucose concentration. Theophylline (T) permitted isoproterenol inhibition of acetate incorporation into lipids (Fig. 6), at the lower rates observed with acetate alone (A) or when lipogenesis from acetate was stimulated by glucose addition (Ag). DISCUSSION
The current observations indicated inhibition of glucose incorporation into lipids by /?-adrenergic agonists plus theophylline was measurable in porcine adipose tissue slices in vitro, as well as in isolated porcine adipocytes (Liu et al., 1989). The inability of this laboratory to measure inhibition of lipogenesis by p-adrenergic agonists (Rule et al., 1989), was a consequence of incubation media composition (no theophylline). It had been demonstrated previously, that isoproterenol inhibited triacylglycerol biosynthesis, but only after the tissue was preincubated for 120min or if this pathway was measured by assessment of palmitate esterification (Rule et al., 1987). Theophylline could exert its permissive effects for B-adrenergic agonist inhibition of lipogenesis, by antagonizing the adenosine receptor (stimulation of the adenosine receptor inhibits adenylate cyclase and CAMP production). An alternative or concomitant
0
g+T
Q
Q+T
A
A+t
Ag Ag+T
Addition Fig. 6. Effect of substrate on p-adrenergic inhibition of lipogenesis. Incubation conditions were as for Fig. 1 except isoproterenol was at 10W6M in all flasks, theophylline was at lo-’ M (T) when present. The radioactive substrates were glucose at either 5 (g) or 20 (G) mM and acetate at 10 mM (A). Radioactive acetate incorporation was measured in the absence (A) or presence (Ag) of 10mM nonradioactive glucose. Data indicated as prnol [U-“C]glucose or [2i4C]acetate incorporated into total lipid per 120 min/g tissue; there were two independent replicates, each with tissue from a different pig; pigs weighed 22.9 and 23.2 kg.
622
HARRY J. MERSMANN
mechanism is inhibition of CAMP phosphodiesterase by theophylline to decrease the catabolism of CAMP. Tissue slices require a greater concentration of theophylline than isolated cells to permit /I-adrenergic inhibition of lipogenesis. This result implies (a) the adenosine receptor is less accessible to theophylline in slices than in cells, (b) the affinity of the adenosine receptor for theophylline increased during cell isolation, (c) isolated cells are more permeable to theophylline than slices, so that phosphodiesterase is more readily inhibited, (d) isolated cell phosphodiesterase has been partially destroyed so there is less to inhibit, or modified so the Ki is lower in cells than slices, (e) combinations of any or all of the above. Modifications in isolated adipocytes relative to tissue fragments have been demonstrated, e.g. rat cells produce more adenosine than slices (Shechter, 1982) and CAMP phosphodiesterase activity is lower in human and rat adipocytes than in tissue fragments (Engfeldt et al., 1985). Differences in metabolism of tissue fragments (or slices or explants) and isolated cells complicate the study of adipose tissue metabolism in vitro, because it is not clear whether the fragment or the isolated cell more closely approximates conditions in uiuo; enzymes used to isolate cells could produce proteolytic changes on the cell surface, so the isolated cell is quite dissimilar to its native state. Adenosine deaminase has a permissive action also for fl-adrenergic inhibition of porcine adipose tissue slice (Fig. 5) and isolated cell (Liu et al., 1989) lipogenesis, but is much less effective than theophylline. If the permissive effect of theophylline is solely via antagonism of the adenosine receptor, adenosine deaminase might be expected to be as effective as theophylline because destruction of the agonist (adenosine) should be as effective as antagonism of the receptor (by theophylline). Previous experience with adenosine and adenosine deaminase in porcine adipose tissue presented another dilemma. Porcine adipose tissue slice lipolytic activity is less responsive to epinephrine in the presence of adenosine and more responsive to epinephrine in the presence of adenosine deaminase (Mersmann, 1984c-Fig. 2), whereas lipogenic rates are not detectably affected by either of these agents (Mersmann and Hu, 1987discussed in text) nor is lipogenesis inhibited by isoproterenol in the presence of adenosine deaminase (Rule er al., 1987). Consequently in the same cell, adenosine concentration appears to control lipolysis to a greater extent than lipogenesis even though the mechanism for adenosine regulation of both pathways is assumed to be through the adenosine receptor, via control of adenylate cyclase and CAMP concentration. Mills and co-workers have confirmed clenbuterol, a P-adrenergic agonist that when fed to pigs reduces fat accretion, is at best a poor agonist for inhibition of lipogenesis (Rule et al., 1987) or stimulation of lipolysis (Mersmann, 1987a) in porcine adipose tissue in vitro. Clenbuterol can be a partial agonist if theophylline or adenosine deaminase is included in the medium (Liu et al., 1989; Liu and Mills, 1989). Similar results are observed by these workers with ractopamine, another /I-adrenergic agonist that reduces fat deposition when fed to pigs. Ractopamine
is a full agonist in the presence of theophylline or adenosine deaminase. Liu and Mills (1989) have demonstrated that clenbuterol and ractopamine both bind very effectively to the fi-adrenergic receptor because each can competitively inhibit the agonist effects of epinephrine under conditions where there is no expression of agonistic activity by either compound, i.e. when no theophylline or adenosine deaminase are present. At this time, it is not apparent whether a compound like clenbuterol or ractopamine stimulates the adipose tissue /I-adrenergic receptor in uiuo or, whether it binds and competitively inhibits the agonistic effects of epinephrine or norepinephrine. This dilemma regarding effects in uiuo has major implications for any projected mechanism of these compounds to reduce fat deposition. This dilemma will be difficult to resolve because of the complexities of establishing mechanism(s) of drug action in uiuo and, in the case of P-adrenergic agonists, to sift through the plethora of both direct and indirect functions controlled by these agonists that could affect adipose tissue metabolism and deposition. Acknowledgements-I
thank C. J. Smith for execution of these experiments; T. W. Acton and J. A. Dague for provision, care and biopsy of the pigs; C. C. Grummert for secretarial assistance. REFERENCES
Choi 0. H., Shamin M. T., Padgett W. L. and Daly J. W. (1988) Caffeine and theophylline analogues: correlation of behavioral effects with activity as adenosine receptor antagonists and as phosphodiesterase inhibitors. Life Sci. 43, 387-398. Engfeldt P., Arner P. and &man J. (1985) Nature of the inhibitory effect of collagenase on phosphodiesterase activity. J. Lipid Res. 26, 977-981. Hu C. Y., Novakofski J. and Mersmann H. J. (1987) Hormonal control of porcine adipose tissue fatty acid release and CAMP concentration. J. Anim. Sci. 64, 1031-1037.
Liu C. Y., Boyer J. L. and Mills S. E. (1989) Acute effects of j-adrenergic agonists on porcine adipocyte metabolism in vitro. J. Anim. Sci. (In press). Liu C. Y. and Mills S. E. (1989) Determination of the affinity of ractopamine or clenbuterol for the betaadrenoceptor of the porcine adipocyte. J. Anim. Sci. (In press). Mersmann H. J. (1983) Effect of anesthetic or analgesic drugs on lipogenic and lipolytic adipose tissue activities. Proc. Sot. exp. Biol. Med. 112, 375-378.
Mersmann H. J. (1984a) Specificity of /?-adrenergic control of lipolysis in swine adipose tissue. Comp. Biochem. Physiol. 77, 39-42.
Mersmann H. J. (1984b) Adrenergic control of lipolysis in swine adipose tissue. Comp. Biochem. Physiol. 11, 43-53. Mersmann H. J. (1984c) Absence of a-adrenergic inhibition of lipolysis in swine adipose tissue. Camp. Biochem. Physiol. 79, 165-170.
Mersmann H. J. (1987a) Acute metabolic effects of adrenergic agents in swine. Am. J. Physiol. 252, E85-E95. Mersmann H. J. (1987b) Primer on beta adrenergic agonists and their effect on the biology of swine. In: The Repartitioning Revolution: Impact of Somatotropin and Beta Adrenergic Agonists on Future Pork Production. Proc.
Univ. of Illinois Pork Ind. Conf. Urbana, IL. Mersmann H. J., Brown L. J., Underwood M. C. and Stanton H. C. (1974) Catecholamine-induced lipolysis in swine. Comp. Biochem. Physiol. 47B, 263-270.
/I-Adrenergic inhibition of lipogenesis Mersmann H. J. and Hu C. Y. (1987) Factors affecting measurements of glucose metabolism and lipolytic rates in porcine adipose tissue slices in vitro. J. Anim. Sci. 64, 148-164. Rule D. C., Smith S. B. and Mersmann H. J. (1987) Effects of adrenergic agonists and insulin on porcine adipose tissue lipid metabolism in vitro. J. Anim. Sci. 65, 136-149. Saggerson E. D. (1985) Hormonal regulation of biosynthetic activities in white adipose tissue. In: New Perspectives in Adipose Tissue: Structure, Function and Development
623
(Edited by Cryer A. and Van R. L. R.), pp. 87-102. Butterworths, London. Shechter Y. (1982) Evaluation of adenosine or related nucleosides as physiological regulators of lipolysis in adipose tissue. Endocrinology 110, 1579-1583. Vernon R. G. and Clegg R. A. (1985) The metabolism of white adipose tissue in vivo and in vitro. In: New Perspectives in Adipose Tissue: Structure, Function and Development (Edited by Cryer A. and Van R. L. R.),
pp. 65568. Butterworths, London.
Camp. Biochem. Physiol.
No. 2, pp. 619-623,
1989
Pergamon Press plc
Printed in Great Britain
INHIBITION LIPOGENESIS
OF PORCINE ADIPOSE TISSUE BY p-ADRENERGIC AGONISTS
HARRY J. MERSMANN US Department of Agriculture, Clay Center, NE 68933 USA* Telephone (7 13) 7987000 (Received 28
July
1989)
Abstract-l. Beta-adrenergic agonists were not effective inhibitors of lipogenesis in porcine adipose tissue slices in vitro; addition of theophylline permitted the inhibition. 2. Inhibition was increased to a greater extent by isoproterenol than epinephrine and was decreased by propranolol, therefore presumably via D-adrenergic receptors. 3. Caffeine, isobutylmethylxanthine and theophylline all permitted inhibition of lipogenesis by /I-adrenergic agonists. 4. It is not clear whether the mechanism for this permissive action is via antagonism of the adenosine receptor, inhibition of CAMP phosphodiesterase or a combination of both. 5. Adenosine deaminase was weakly permissive, presumably through destruction of adenosine. Inhibition of lipogenesis was observed with glucose or acetate as lipogenic substrate and in the presence
or absence of albumin.
INTRODUCTION
adipose tissue lipid synthesis (lipogenesis) is inhibited (Saggerson, 1985) and lipid degradation (lipolysis) is stimulated (Vernon and Clegg, 1985) in vitro by B-adrenergic agonists. Initial studies by this investigator (Mersmann et al., 1974) established that p-adrenergic agonists increase rates of lipolysis in porcine adipose tissue in vitro. Subsequently, it was shown that this tissue responds to a select number of agonists only; it was concluded that the receptor on this tissue exhibits stringent specificity for control of lipolysis and tissue CAMP concentration (Mersmann, 1984a,b; Hu et al., 1987). The specificity can be demonstrated in vivo also (Mersmann, 1987a). Evidence was presented to indicate /3-adrenergic agonists alone, including isoproterenol, do not inhibit lipogenesis in porcine adipose tissue in vitro (Rule et al.. 1987). More direct assessment of aspects of triacylglycerol biosynthesis indicated isoproterenol inhibits but only under specific incubation conditions (Rule et al., 1987). Recently Mills and co-workers (Liu et al., 1989; Liu and Mills, 1989) have confirmed that epinephrine alone is a poor agonist for inhibition of lipogenesis when incubated with isolated porcine adipocytes in vitro. However, if the incubation medium contains theophylline or adenosine deaminase, epinephrine effectively inhibits lipogenesis. The purpose of the studies indicated herein was to determine if observations similar to those of Mammalian
*Agricultural Research Service, Roman L. Hruska, US Meat Animal Research Center. P.O. Box 166. Clav Center, NE, USA. Mention of trade names, proprietary products or specific equipment 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.
Mills and co-workers could be demonstrated in tissues lices (the preparation used in this laboratory) or whether /I-adrenergic agonist inhibition of lipogenesis was observable only after exposure to the proteolytic enzymes used for adipocyte isolation. METHODS Crossbred (l/4 Yorkshire, l/4 Large White, I/4 Chester White, l/4 Landrace) castrated male pigs (Sus domesticus), were used at weights between 20 and 36 kg. Pigs were about 9-15 wk of age and were fed a corn-soybean meal diet containing 18% calculated protein ad libitum,from weaning at 4wk. Adipose tissue biopsies were obtained from the dorsal neck region of pigs anesthetized with pentothal Na, as indicated previously (Mersmann, 1983). Tissue was transported to the laboratory in 0.9% NaCl, 5.6 mM glucose, 25 mM Hepes at pH 7.4 and 35°C. Tissue from an individual animal was used to prepare a pool of 0.4 mm slices with a tissue slicer. Slices from the pool were used to execute an individual replicate of an experiment. Lipogenesis was assessed by measuring incorporation of i4C-glucose into a total lipid extract. The incubation medium contained Krebs-Ringer bicarbonate buffer with 50% of the indicated calcium concentration, 5 mM glucose, 0.5 FCi radioactive glucose, 0.57 mM ascorbate, 1munit insulin/ml and 100 mg tissue slices, unless indicated otherwise. Incubations were in triplicate at 37°C for 120 min, with reciprocal shaking at 90 strokes/min. Reactions were stopped by injection of 0.25 ml 1 N H,SO,; lipids were extracted with chloroform: methanol (2 : 1, v/v); radioactivity in the extract was quantified by liquid scintillation spectrometry. Details of these procedures were reported previously (Mersmann and Hu, 1987). In one experiment, 0.5pCi i4C-acetate was used as substrate. Additions to incubation flasks and modifications to the standard incubation conditions are detailed for each experiment. Isoproterenol bitartrate (12760), theophylline (T’1633), deaminase (A6648), adenosine (A925 I), adenosine (-)epinephrine bitartrate (E4375), 3-isobutyl-l-methylxanthine (15879), caffeine (CO750), m-propranolol HCl (P0884) were purchased from Sigma Chemical Co., St Louis, MO 63178, USA. Bovine serum albumin, Bovuminar
619
HARRYJ. MERSMANN
620
reagent CRG-7 (2295-00) was purchased from Armour Pharmaceutical Co., Kankakee, IL 60901, USA. Crystalline porcine insulin (SI-144-2A; 24.7 units/mg) was generously provided by Lilly Research Laboratory, Greenfield, IN 46140, USA. D-[‘4c(u)]ghCOSe (NEC-042X) and sodium [2-V] acetate (NEC085H) were purchased from NEN Research Products, DuPont Company, Wilmington, DE, 19898, USA.
$
10
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c e
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0”
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1
2
RESULTS
The first attempt to replicate the observations of Mills and co-workers involved incubation of tissue slices with epinephrine or epinephrine plus 5 x 10m4M theophylline. There was a tendency for glucose incorporation into both CO, and lipid to increase slightly with increasing epinephrine concentration, whereas in the presence of theophylline there was a tendency for incorporation to decrease with increased epinephrine concentration (data not indicated). This result qualitatively reproduced the observations of Mills and co-workers (Liu et al., 1989), in that lipogenesis was inhibited, albeit to a small extent only, by a /?-adrenergic agonist, epinephrine; theophylline was a necessary medium component for this inhibition. It reproduced the observations of Rule et al. (1987) also, in that there was no inhibition and possibly slight stimulation when theophylline was not in the incubation medium. Epinephrine, an a- plus fi-adrenergic agonist, was compared with isoproterenol, a pure /I-adrenergic agonist; both agonists were studied in the presence of 5 x 10e4 M theophylline (Fig. 1). Epinephrine and isoproterenol each tended to inhibit lipogenesis, with isoproterenol being more potent than epinephrine. The inhibition was only about 30% at the highest concentrations of either agonist. All subsequent experiments used isoproterenol to eliminate the possibility of a-adrenergic effects when utilizing the mixed agonist, epinephrine. Theophylline (5 x 10m4M) alone, i.e. no isoproterenol, did not inhibit insulin-stimulated lipogenesis; glucose incorporation into lipids was 5.94 f 0.18 (3 experiments) in the control flasks and 5.64 f 0.90 in the presence of 5 x 10e4 M theophylline. Liu et al.
0
0.01
0.1
0.6
Theophylline,
1
6
ImMl
Fig. 2. Theophylline saturation. Incubation conditions and data presentation were the same as for Fig. 1 except isoproterenol was at 10m6M and theophylline concentration was varied as indicated. There were two independent replications, each with tissue from a different pig; pigs weighed 19.3 and 22.7 kg.
(1989) indicated that, in the presence of insulin, theophylline alone did not inhibit lipogenesis. Because the magnitude of inhibition of tissue slice lipogenesis by isoproterenol (or epinephrine) was small in the presence of 5 x 10m4M theophylline, the concentration of theophylline was titrated at a fixed concentration ( lO-6 M) of isoproterenol (Fig. 2). Lipogenesis was strongly inhibited by isoproterenol but only at theophylline concentrations >5 x low4 M. These concentrations were greater than are necessary to demonstrate similar effects in isolated porcine cells (Liu et al., 1989). Propranolol, a fi-adrenergic antagonist, reversed the inhibition of lipogenesis by isoproterenol in the presence of theophylline (Fig. 3). Because isoproterenol was more potent than epinephrine and isoproterenol-stimulated inhibition was reversed by propranolol, a pure j?-adrenergic agonist or antagonist, respectively, the inhibition of lipogenesis was almost certainly through the fi-adrenergic receptor. At a medium concentration of lo-‘M, theophylline (T) and the related compound, caffeine (Caf), both permitted the inhibition of lipogenesis by isoproterenol to the same extent (Fig. 4) whereas
14 8
12
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10
tI:
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6
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4 2 0
0 -6
Agonirt,
log [MI
Fig. 1. Inhibition of lipogenesis by epinephrine and isoproterenol. Tissue slices were incubated with 5 mM glucose, 0.57 mM ascorbate, 5 x 1O-4 M theophylline, 1 m unit insulin/ml and epinephrine (hatched bars) or isoproterenol (solid bars) concentrations between lo-* and 10p4M, as indicated. Lipogenesis is expressed as pmol [UJ4C]glucose incorporated into total lipid per 120 min/g tissue. Data are mean with SD indicated as error bars. There were three independent replications, each with tissue from a different pig; pigs weighed 21.8 f 0.5 kg.
-8
-7
Propranolol,
-6
-5
log
-4
[Ml
Fig. 3. Reversal of /I-adrenergic stimulated inhibition of lipogenesis by a fi-adrenergic antagonist. Incubation conditions and data presentation were the same as for Fig. 1, except there was 10w6M isoproterenol and lo-)M theophylline were present in all flasks; propranolol was varied as indicated. There were two independent replications each with tissue from a different pig; pigs weighed 28.7 and 36.0 kg. The uninhibited lipogenic rate [indicated as mean (SD)] was 7.8 (2.3) prnol [II-“‘Clglucose incorporated into total lipid per 120 min/g tissue (10m6M isoproterenol and no theophylline in the Rasks).
/I-Adrenergic inhibition of lipogenesis
8
I
621
120 #
100 80
_
8 P 2
60
8 J 2
g b 8
40 2o
g $ P
C
1
IBX
Cat
Ad)
Ad
Al
60 60 40 20
0
Al+t
Addition
(IBX), a more potent
as
IO
100
9as
Adenosina Deaminase,
Fig. 4. Miscellaneous effects on isoproterenol inhibition of lipogenesis. Incubation conditions generally were the same as for Fig. 1. Isoproterenol was at lO-‘jM in all flasks including the controls (C); theophylline (T), methylisobutyl xanthine (IBX) and caffeine (Caf) were at lo-)M [theophylline was present only where indicated (T)]; adenosine deaminase (AdD) was at 1 unit/ml; adenosine (Ad) was at IO-‘M; bovine serum albumin (Al) was at 30mg/ml or 3%. Data are indicated as percent of control (C) calculated on an individual animal basis because of divergent control values (8.46 and 4.34 pmol [U-r4C]glucose incorporated into total lipid per 120 min/g tissue); mean with SD indicated as error bars. There were two independent replications each with tissue from a different pig; pigs weighed 29.3 and 32.1 kg.
isobutylmethylxanthine
100
adeno-
sine receptor antagonist and inhibitor of CAMP phosphodiesterase than theophylline or caffeine (Choi et al., 1988), permitted greater inhibition of lipogenesis by isoproterenol. Albumin is used in media to assess lipogenesis by some investigators (see Mersmann and Hu, 1987, for references) and is a necessary medium component in studies with isolated adipocytes to maintain cell integrity. In the presence of albumin, lipogenesis might be indirectly inhibited by isoproterenol, because isoproterenol-stimulated lipolysis would produce free-fatty acids that could cause feedback inhibition of lipogenesis. This was not the case (Fig. 4Al), as was observed previously (Rule et al., 1987Table 1). Isoproterenol inhibited lipogenesis in the presence of albumin, only when theophylline was present (Fig. 4-Al+ T). Addition of lo-’ M adenosine (Ad) had little effect on the isoproterenol response in the absence of theophylline; addition of 1 unit of adenosine deaminase/ ml (AdD) to the isoproterenol-containing medium (no theophylline), tended to permit the inhibition (Fig. 4). Titration of adenosine deaminase concentration, indicated the permission of isoproterenol inhibition of lipogenesis was adenosine deaminase concentration dependent (Fig. 5); however, the maximum inhibition was only about 30% even at extremely high concentrations of the enzyme. Initial studies suggested, that not only was lipogenesis inhibited by epinephrine (in the presence of theophylline), but incorporation of glucose carbon into CO, was inhibited, also. Experiments in this manuscript used 5 mM glucose to mimic the conditions of Mills and co-workers; theophylline permitted isoproterenol-stimulated inhibition of lipogenesis with both 5 (g) or 20 (G) mM glucose as lipogenic substrate (Fig. 6), thus, the inhibition observed by Mills, but not previously by this laboratory (routine use of 20mM glucose), was not the
1000
mu/ml
Fig. 5. Saturation of adenosine deaminase. Incubation conditions were the same as for Fig. 1 except isoproterenol was at 10m6M and there was no theophylline present. Adenosine deaminase was varied as indicated. These were the same two pigs as in Fig. 3.
result of glucose concentration. Theophylline (T) permitted isoproterenol inhibition of acetate incorporation into lipids (Fig. 6), at the lower rates observed with acetate alone (A) or when lipogenesis from acetate was stimulated by glucose addition (Ag). DISCUSSION
The current observations indicated inhibition of glucose incorporation into lipids by /?-adrenergic agonists plus theophylline was measurable in porcine adipose tissue slices in vitro, as well as in isolated porcine adipocytes (Liu et al., 1989). The inability of this laboratory to measure inhibition of lipogenesis by p-adrenergic agonists (Rule et al., 1989), was a consequence of incubation media composition (no theophylline). It had been demonstrated previously, that isoproterenol inhibited triacylglycerol biosynthesis, but only after the tissue was preincubated for 120min or if this pathway was measured by assessment of palmitate esterification (Rule et al., 1987). Theophylline could exert its permissive effects for B-adrenergic agonist inhibition of lipogenesis, by antagonizing the adenosine receptor (stimulation of the adenosine receptor inhibits adenylate cyclase and CAMP production). An alternative or concomitant
0
g+T
Q
Q+T
A
A+t
Ag Ag+T
Addition Fig. 6. Effect of substrate on p-adrenergic inhibition of lipogenesis. Incubation conditions were as for Fig. 1 except isoproterenol was at 10W6M in all flasks, theophylline was at lo-’ M (T) when present. The radioactive substrates were glucose at either 5 (g) or 20 (G) mM and acetate at 10 mM (A). Radioactive acetate incorporation was measured in the absence (A) or presence (Ag) of 10mM nonradioactive glucose. Data indicated as prnol [U-“C]glucose or [2i4C]acetate incorporated into total lipid per 120 min/g tissue; there were two independent replicates, each with tissue from a different pig; pigs weighed 22.9 and 23.2 kg.
622
HARRY J. MERSMANN
mechanism is inhibition of CAMP phosphodiesterase by theophylline to decrease the catabolism of CAMP. Tissue slices require a greater concentration of theophylline than isolated cells to permit /I-adrenergic inhibition of lipogenesis. This result implies (a) the adenosine receptor is less accessible to theophylline in slices than in cells, (b) the affinity of the adenosine receptor for theophylline increased during cell isolation, (c) isolated cells are more permeable to theophylline than slices, so that phosphodiesterase is more readily inhibited, (d) isolated cell phosphodiesterase has been partially destroyed so there is less to inhibit, or modified so the Ki is lower in cells than slices, (e) combinations of any or all of the above. Modifications in isolated adipocytes relative to tissue fragments have been demonstrated, e.g. rat cells produce more adenosine than slices (Shechter, 1982) and CAMP phosphodiesterase activity is lower in human and rat adipocytes than in tissue fragments (Engfeldt et al., 1985). Differences in metabolism of tissue fragments (or slices or explants) and isolated cells complicate the study of adipose tissue metabolism in vitro, because it is not clear whether the fragment or the isolated cell more closely approximates conditions in uiuo; enzymes used to isolate cells could produce proteolytic changes on the cell surface, so the isolated cell is quite dissimilar to its native state. Adenosine deaminase has a permissive action also for fl-adrenergic inhibition of porcine adipose tissue slice (Fig. 5) and isolated cell (Liu et al., 1989) lipogenesis, but is much less effective than theophylline. If the permissive effect of theophylline is solely via antagonism of the adenosine receptor, adenosine deaminase might be expected to be as effective as theophylline because destruction of the agonist (adenosine) should be as effective as antagonism of the receptor (by theophylline). Previous experience with adenosine and adenosine deaminase in porcine adipose tissue presented another dilemma. Porcine adipose tissue slice lipolytic activity is less responsive to epinephrine in the presence of adenosine and more responsive to epinephrine in the presence of adenosine deaminase (Mersmann, 1984c-Fig. 2), whereas lipogenic rates are not detectably affected by either of these agents (Mersmann and Hu, 1987discussed in text) nor is lipogenesis inhibited by isoproterenol in the presence of adenosine deaminase (Rule er al., 1987). Consequently in the same cell, adenosine concentration appears to control lipolysis to a greater extent than lipogenesis even though the mechanism for adenosine regulation of both pathways is assumed to be through the adenosine receptor, via control of adenylate cyclase and CAMP concentration. Mills and co-workers have confirmed clenbuterol, a P-adrenergic agonist that when fed to pigs reduces fat accretion, is at best a poor agonist for inhibition of lipogenesis (Rule et al., 1987) or stimulation of lipolysis (Mersmann, 1987a) in porcine adipose tissue in vitro. Clenbuterol can be a partial agonist if theophylline or adenosine deaminase is included in the medium (Liu et al., 1989; Liu and Mills, 1989). Similar results are observed by these workers with ractopamine, another /I-adrenergic agonist that reduces fat deposition when fed to pigs. Ractopamine
is a full agonist in the presence of theophylline or adenosine deaminase. Liu and Mills (1989) have demonstrated that clenbuterol and ractopamine both bind very effectively to the fi-adrenergic receptor because each can competitively inhibit the agonist effects of epinephrine under conditions where there is no expression of agonistic activity by either compound, i.e. when no theophylline or adenosine deaminase are present. At this time, it is not apparent whether a compound like clenbuterol or ractopamine stimulates the adipose tissue /I-adrenergic receptor in uiuo or, whether it binds and competitively inhibits the agonistic effects of epinephrine or norepinephrine. This dilemma regarding effects in uiuo has major implications for any projected mechanism of these compounds to reduce fat deposition. This dilemma will be difficult to resolve because of the complexities of establishing mechanism(s) of drug action in uiuo and, in the case of P-adrenergic agonists, to sift through the plethora of both direct and indirect functions controlled by these agonists that could affect adipose tissue metabolism and deposition. Acknowledgements-I
thank C. J. Smith for execution of these experiments; T. W. Acton and J. A. Dague for provision, care and biopsy of the pigs; C. C. Grummert for secretarial assistance. REFERENCES
Choi 0. H., Shamin M. T., Padgett W. L. and Daly J. W. (1988) Caffeine and theophylline analogues: correlation of behavioral effects with activity as adenosine receptor antagonists and as phosphodiesterase inhibitors. Life Sci. 43, 387-398. Engfeldt P., Arner P. and &man J. (1985) Nature of the inhibitory effect of collagenase on phosphodiesterase activity. J. Lipid Res. 26, 977-981. Hu C. Y., Novakofski J. and Mersmann H. J. (1987) Hormonal control of porcine adipose tissue fatty acid release and CAMP concentration. J. Anim. Sci. 64, 1031-1037.
Liu C. Y., Boyer J. L. and Mills S. E. (1989) Acute effects of j-adrenergic agonists on porcine adipocyte metabolism in vitro. J. Anim. Sci. (In press). Liu C. Y. and Mills S. E. (1989) Determination of the affinity of ractopamine or clenbuterol for the betaadrenoceptor of the porcine adipocyte. J. Anim. Sci. (In press). Mersmann H. J. (1983) Effect of anesthetic or analgesic drugs on lipogenic and lipolytic adipose tissue activities. Proc. Sot. exp. Biol. Med. 112, 375-378.
Mersmann H. J. (1984a) Specificity of /?-adrenergic control of lipolysis in swine adipose tissue. Comp. Biochem. Physiol. 77, 39-42.
Mersmann H. J. (1984b) Adrenergic control of lipolysis in swine adipose tissue. Comp. Biochem. Physiol. 11, 43-53. Mersmann H. J. (1984c) Absence of a-adrenergic inhibition of lipolysis in swine adipose tissue. Camp. Biochem. Physiol. 79, 165-170.
Mersmann H. J. (1987a) Acute metabolic effects of adrenergic agents in swine. Am. J. Physiol. 252, E85-E95. Mersmann H. J. (1987b) Primer on beta adrenergic agonists and their effect on the biology of swine. In: The Repartitioning Revolution: Impact of Somatotropin and Beta Adrenergic Agonists on Future Pork Production. Proc.
Univ. of Illinois Pork Ind. Conf. Urbana, IL. Mersmann H. J., Brown L. J., Underwood M. C. and Stanton H. C. (1974) Catecholamine-induced lipolysis in swine. Comp. Biochem. Physiol. 47B, 263-270.
/I-Adrenergic inhibition of lipogenesis Mersmann H. J. and Hu C. Y. (1987) Factors affecting measurements of glucose metabolism and lipolytic rates in porcine adipose tissue slices in vitro. J. Anim. Sci. 64, 148-164. Rule D. C., Smith S. B. and Mersmann H. J. (1987) Effects of adrenergic agonists and insulin on porcine adipose tissue lipid metabolism in vitro. J. Anim. Sci. 65, 136-149. Saggerson E. D. (1985) Hormonal regulation of biosynthetic activities in white adipose tissue. In: New Perspectives in Adipose Tissue: Structure, Function and Development
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(Edited by Cryer A. and Van R. L. R.), pp. 87-102. Butterworths, London. Shechter Y. (1982) Evaluation of adenosine or related nucleosides as physiological regulators of lipolysis in adipose tissue. Endocrinology 110, 1579-1583. Vernon R. G. and Clegg R. A. (1985) The metabolism of white adipose tissue in vivo and in vitro. In: New Perspectives in Adipose Tissue: Structure, Function and Development (Edited by Cryer A. and Van R. L. R.),
pp. 65568. Butterworths, London.