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Growth hormone increases the lipolytic sensitivity for catecholamines in adipocytes from healthy adults
Life Sciences, %'ol.54, No. 18, pp. 1335-1341, 1994 Copyright© 1994 Elsevier Science Ltd Printed in the USA. All fights reserved 0024-3205/94 $6.00 + .00
Pergamon
G R O W T H H O R M O N E I N C R E A S E S THE LIPOLYTIC SENSITIVITY FOR C A T E C H O L A M I N E S IN A D I P O C Y T E S F R O M H E A L T H Y ADULTS Claude Marcus, Per Bolme, Gunilla Micha-Johansson Veronique Margery and Mikael Bronneg&rd Department of Pediatrics, Huddinge University Hospital, Karolinska Institute, Stockholm, Sweden. (Received
in final form
February
14, 1994)
Summary The lipolytic effect of growth hormone (GH) was investigated in adipocytes obtained during elective surgery from otherwise healthy adults, 18-40 years old. No lipolytic or antilipolytic effect of GH was found when the cells were incubated with GH alone during 30min-6h. When the cells were preincubated with GH during 3h, the lipolytic sensitivity for isoprenaline increased markedly without any change in maximal lipolysis. However, a full effect was only obtained if GH was also present during the incubation with isoprenaline. GH did not alter DB-CAMP, enprophylline, or forskolineinduced lipolysis in human fat cells. In conclusion, GH had no direct lipolytic effect on human fat cells but GH markedly increased the catecholamine sensitivity. The site of the GH effect seems to be in the i3-adrenoceptors or in the G, coupling protein.
Key Words: adipocytcs, lipolysis, glycerol, growth hormone, catccholaraincs It is well established that growth hormone (GH) is important not only for linear growth but also for adipocyte differentiation and energy metabolism in both animals and man. GH treatment of GH-deficient adults reduces the abdominal fat mass and decreases fat cell size (1,2). How these effects of GH on adipocyte cells are mediated is not yet fully understood. It seems to be established that GH reduces glucose uptake in the fat cells of humans (1,3) and rats (4), both basal and insulin-stimulated. GH weakly stimulates basal lipolysis in rat fat cells (5) and strongly stimulates theophyllamine and catecholamine lipolysis (6). In 3T3-F442A cells the lipolytic effect of GH seems to be due to a direct effect on the lipase (7). However, the acute effect of GH on lipolysis in human fat cells remains unclear. Incubation with GH did not affect lipolysis in human fat cells in culture (8) and GH treatment does not increase noradrenaline induced lipolysis in isolated abdominal and gluteal adipocytes from GH deficient children (1). In vivo, the lipolytic effect of GH in humans has been demonstrated both in adults and children using various methodological Adress correspondence to Dr Claude Marcus, Pediatric Endocrine Research Unit, Huddinge Hospital S-141 46 Huddinge, Sweden
1336
Lipolytic Effect of GH in Adipocytes
Vol. 54, No. 18, 1994
aproaches (9-11). This may indicate that the lipolytic effects of GH are mediated in different ways in humans than in other species. The aim of the present study was to reevaluate the effects of GH on basal lipolysis and lipolysis stimulated by both catecholamines and agents which stimulate lipolysis via activation of the lipolytic cascade at levels below the 6-adrenergic receptors in isolated adipocytes from healthy adults.
Material and methods
Abdominal subcutaneous adipose tissue was obtained during routine cholecystectomy and inguinal hernia operations on 17 non-obese patients, 12 males and 5 females, 18-40 years old. They were otherwise healthy, of normal weight and not on medication. After an overnight fast, general anesthesia was induced with thiopental sodium and maintained with a mixture of oxygen and nitrous oxide. The study was approved by the Ethics Committee of Karolinska Institute. Adipose tissue was removed from the surgical incision at the beginning of the operation. It was cut into fragments and isolated from the stroma by incubation with collagenase for 45 minutes in Krebs Ringer phosphate (KRP) buffer, pH 7.4, containing 4% bovine serum albumin (BSA), mainly using Rodbell's method (12). The adipocytes were washed in KRP albumin buffer and aggregated material was removed by filtration through a silk cloth. Lipolysis experiments. Fat cell suspensions (2%, vol/vol) were incubated in duplicate in KRP buffer (ph 7.4) with 40 mg/ml of BSA, 0.1 mg/ml of ascorbic acid and 1 mg/ml of glucose at 37°C for 30 min-6h. At the end of the incubations an aliquot of the medium was removed for determination of the glycerol release which was used as an index of lipolysis. In a subset of experiments, cells were preincubated with GH for 1-3h and then preincubated without GH for 0-2h as shown in Table 1. Thereafter and when the cells were between preincubations, they were washed twice with fresh KRP-albumin buffer and reincubated during the fourth hour with or without GH and isoprenaline in concentrations of 0, 10-13, 10-11 (submaximal concentrations) and 10 .8 mol/I (i.e., the isoprenaline concentration in our incubation system needed to obtain maximal lipolysis). In control experiments, the cells were treated in exactly the same way, but without GH. The glycerol concentration was analyzed by a sensitive kinetic bioluminescence method (13). Adenosine, secreted from fat cells, is a potent inhibitor of lipolysis. We have previously shown that the adenosine contamination is of no importance in this incubation system (14). All experiments were obtained on single adipose tissue samples. Chemicals. Recombinant GH was a gift from Kabi Pharmacia, Stockholm, Sweden. The collagenase was prepared from Clostridium histolyticum and was of Sigma type 1. Dialyzed BSA (fraction V) was purchased from the Armour Pharmaceutical Company, Eastbourne, England. The same batches of collagenase and albumin were used in all experiments. Isoprenaline, forskolin, enprophylline, dibutyryl cAMP and cycIoheximide came from Sigma Chemical Company, St. Louis, MO, USA. Statistics and analysis of results. The Student's paired and unpaired t-tests were used when appropriate.
Vol 54, No. 18, 1994
Lipolytic Effect of GH in Adipocytes
1337
Results Effects of GH on basal lipolysis. Adipocytes from three patients were incubated for 30min6h in the presence or absence of GH (7ng/ml). Lipolysis was constant for 6h - i.e., the glycerol concentration increased linearly (data not shown). No lipolytic or antilipolytic effect of GH was observed and the glycerol time-curves were identical, whether or not GH was present in the incubation medium.
Table
1
Effect of GH, during various incubation times, on lipolysis induced by submaximal isoprenaline concentrations Incubation hour 1 2 3 + + + +
+
+
+
+
+
+
lipolytic effect 0 0 (+) +
-
+
(+)
+
4 -
p
ns <0.05 ns
A d i p o c y t e s w e r e i n c u b a t e d d u r i n g four hours w i t h (+) or without (-) GH and, d u r i n g the f o u r t h hour, w i t h isoprenaline (I0-ii and 10-13 mol/l) as well. The l i p o l y t i c e f f e c t of i s o p r e n a l i n e alone, i° e., w i t h o u t GH, w h e r e the cells w e r e h a n d l e d the same way, was determined as control experiments, n=4, Lipolysis denotes the effect of GH compared to control e x p e r i m e n t s , ns = not s i g n i f i c a n t .
GH effect on isoprenaline-induced lipolysis. In adipocytes from four subjects, the effect on lipolysis of preincubation with GH (7ng/ml), induced by various concentrations of isoprenaline, was investigated, as described in Material and Methods and in Table 1. GH did not affect the maximal isoprenaline-induced lipolysis (Fig.l). However, the lipolytic effect of submaximal concentrations of isoprenaline was markedly enhanced by preincubation with GH for 3h. The effect of GH was significant only if GH was present in the incubation medium both during the whole preincubation period and during the incubation with isoprenaline (Table 1, Fig. 1). The effect of cycloheximide (protein synthesis inhibitor) on GH effets was investigated in seven patients. The effects of cycloheximide varied markedly between individuals. In three experiments cycloheximide markedly reduced the effect of GH on isoprenaline induced lipolysis without affecting the lipolysis induced by isoprenaline alone but in in three experiments no effect was observed and the overall effect of cycloheximide was not significant.
1338
Lipolytic Effect of GH in Adipocytes
Vol. 54, No. 18, 1994
A
Z0
100
B
VISO • ISO+GH • ISO+GH
/
i00 / '
75
////50 "J~""
50 H
25
'~////////
75
//I/ 25
q4 0 dO
0
-13
-ii Log
0
-8
Isoprenaline
-13
-ii
-8
Concentration
Fi.q. 1 The e f f e c t of GH on l i p o l y s i s i n d u c e d by i s o p r e n a l i n e . H u m a n a d i p o c y t e s w e r e p r e i n c u b a t e d for 3h w i t h (filled triangles) or w i t h o u t (open triangles) GH (7ng/ml) and thereafter washed and reincubated for lh with isoprenaline in the c o n c e n t r a t i o n s indicated (iso) alone (A,) or in c o m b i n a t i o n w i t h GH (B, filled triangles). * = p<0.05, Data p r e s e n t e d are m e a n s ± S D of four e x p e r i m e n t s m a d e in d u p l i c a t e .
Effect of GH on lipo!ysis induced by a.qents actin.q at postreceptor sites. In another set of experiments with adipocytes from four subjects, the lipolytic effects of enprophylline (phosphodiesterase inhibitor(15)), dibutyryI-CAMP (stable CAMP analogue) and forskolin (aclenylate cyclase activator (16)) were investigated either after preincubation with GH for 3h and with GH in the incubation medium or without GH. Adipocytes from the same subjects were also incubated with isoprenaline +GH, as previously described. Although that GH stimulated isoprenaline induced lipolysis significantly (p<0.05), the dose-response curves ±GH with agents stimulating lipolysis at a postreceptor level were identical (Fig.2).
Discussion In the present study we have shown that, in abdominal adipocytes from healthy adults, GH increases catecholamine lipolytic sensitivity without affecting the maximal effect induced by the drug. The effect of GH was seen after 3h preincubation, which is in agreement with previous in vivo results in humans (9,10) and with results from studies of
Vol. 54, No. 18, 1994
Lipolytic Effect of GH in Adipoeytes
1339
rat adipocytes (5,6). We were not able to demonstrate any direct lipolytic effect of GH in human adipocytes. This is in contrast to what has been reported in rat adipocytes and in 3T3 F442A, cells where GH has a direct stimulatory effect on lipolysis (5,7) and also increases the maximal effect of various agents such as theophylline and catecholamines (6,7). It has previously been reported that there is neither any lipolytic effect of GH in rabbits in vivo (17) nor in vitro (18). Our results are further indications of marked differences between species as regards the lipolytic effect of GH.
1.5
O ISO Q ISO+GH
1.5
O EHPRO • ENPRO+GH [~ FORSKOLIN m FORSKOLIN+GH
1.0
O o~ ~ ~0.5
0.5
0
-113
111
' -8
-'5 -'4
' -3
o
'
Log Drug Concentration,
-'9
-'8
"~
-'6
-'5
"4
'3
mol/l
The lipolytic effect of agents a c t i n g at p o s t r e c e p t o r sites with or w i t h o u t GH. Human adipocytes were p r e i n c u b a t e d for 3h w i t h GH and t h e r e a f t e r w a s h e d and reincubated for lh w i t h GH in combination with isoprenaline (iso), forskolin, dibutyryl-CAMP or e n p r o p h y l l i n e in the c o n c e n t r a t i o n s i n d i c a t e d (filled symbols). The cells were also preincubated and i n c u b a t e d w i t h o u t GH (open symbols) p r e s e n t in the i n c u b a t i o n medium.
In rat adipocytes the lipolytic effect of GH is depressed by cycloheximide which indicates that protein synthesis is involved (5). In the present study, the potentiating effect of GH on catecholamine induced lipolysis was partly depressed by cycloheximide only in some experiments. GH is secreted in pulses and it is possible that variations in the GH secretion pattern during the time period prior to the fat biopsy may explain the varying effect of cycloheximide. However, it is likely that GH influences lipolysis in more than one way as previously has been suggested (19). In weanling rats GH induces lipolysis without any lag period (20) and the same is observed in diabetic rats (21). Furthermore, in the present study, GH had to be present both during the whole preincubation period and the isoprenaline incubation period to produce the full effect, which also may indicate that GH
1340
Lipolytic Effect of GH in Adipocytes
Vol. 54, No. 18, 1994
has more effect on the lipolysis-promoting system than protein synthesis stimulation alone. GH failed to induce any increase in the lipolytic effect of forskolin, dibutyryl-CAMP or enprophyllin. All these agents activate lipolysis at a level below the 13-adrenergic receptors and the G s protein. Thus, it is likely that the effect of GH is due to changes in 13adrenoceptor number or functional coupling to the G s protein. This is well in accordance with the observation that GH increases sensitivity but not the maximal response of isoprenaline. It has previously been shown that there are spare g-adrenoceptors on human fat cells (22). Thus, an increased number of functionally coupled receptors, would only increase the sensitivity.
Acknowled,qements This study was supported by grants from the Swedish Society of Medicine, the Swedish Medical Research Council (19X-9941), Karolinska Institute, Harald and Greta Jeansson, the Golje Memorial, The Crown Princess Lovisa/Thileman, the Swedish Diabetes, the General Maternity Hospital, the Nordic Insulin and the Eriksson Research Foundations.
References 1. M. ROSENBAUM, J. GERTNER and R. LEIBEL. J Clin Endocrinol Metab. 6._9_912741281 (1989). 2. R. J. MARTIN, M. DREWRY, D. JEWELL, R. HARRIS, R. YOUNG and J. S. PATTON. Int J Obesity 1.__33327-335, (1989). 3. G. NYBERG, S. BOSTROM, R. JOHANSSON and U SMITH. Acta Endocrinologica 9_.55 129-133, (1980). 4. B. MALOFF, J. LEVINE and D. LOCKWOOD. Endocrinology 107:538-544 (1980). 5. A.B. CALDWELL and J.N. FAIN. Horm Metab Res 23-5, (1970). 6. M GOODMAN. Endocrinology 114131-135, (1984). 7. J. DIETZ and J. SCHWARTZ. Metabolism, 40800-806, (1991). 8. G. NYBERG and U. SMITH. Horm Metab Res 9:22-27, (1977). 9. P. METCALFE, D.G. JOHNSTON, R. NOSADINI, H. 0RSKOV, K. G. ALBERTI. Diabetologia 2__0123-128, (1981). 10. G. VAN VLIET, D. BOSSON, M. CRAEN, M. DU CAJU, P. MALVAUX and M. VANDERSCHUEREN-LODEWEYCKS. J Clin Endocrinol Metab 6._.55876-879 (1987). 11. P. BOYLE, A. AVOGARO, L. SMITH, D. BIER, A. PAPPU, R. ILLINGWORTH AND P. CRYER. Am J. Physiol. 263 E168-E172, (1992). 12. M. RODBELL. J. Biol. Chem. 239375-80 (1964). 13. J. HELLMER, P. ARNER AND A LUNDIN Analythical Biochem 177132-37 (1989). 14. C. MARCUS, B. KARPE, P. BOLME, T. SONNENFELD AND P. ARNER. J. Clin. Invest. 7.__991812-18(1987). 15. B. FREDHOLM and E. LINDGREN Acta Pharmacol. Toxicol. 5.~46469 (1983). 16. K. SEAMON, W. PADGETT, and J. DALY. Proc. Natl. Acad. Sci. 7._883363-3367, (1981). 17. J. KNUDTZON, P.D. EDMINSON, K.L. REICHELT. Hormone Res 2110-18, (1985). 18. C. BOWDEN, K. WHITE, U. LEWIS and G. TUTWEILER. Metabolism. 4237-243, (1985).
Vol. 54, No. 18, 1994
Lipolytic Effect of GH in Adipocytes
1341
19. H. M. GOODMAN. Endocrinology, 109, 120-129, (1981). 20. H.M. GOODMAN, V. CORIO. Endocrinology 109 2046-2053, (1981). 21. S. SOLOMON, S. D. SIBLEY, T. M. CUNNINGHAM. Endocrinology 127., 1544-46, (1990). 22. P. ARNER, J. HELLMER, A. WENNLUND, J. OSTMAN and P. ENGFELDT. Eur J Pharm 146 45-56 (1983).
Pergamon
G R O W T H H O R M O N E I N C R E A S E S THE LIPOLYTIC SENSITIVITY FOR C A T E C H O L A M I N E S IN A D I P O C Y T E S F R O M H E A L T H Y ADULTS Claude Marcus, Per Bolme, Gunilla Micha-Johansson Veronique Margery and Mikael Bronneg&rd Department of Pediatrics, Huddinge University Hospital, Karolinska Institute, Stockholm, Sweden. (Received
in final form
February
14, 1994)
Summary The lipolytic effect of growth hormone (GH) was investigated in adipocytes obtained during elective surgery from otherwise healthy adults, 18-40 years old. No lipolytic or antilipolytic effect of GH was found when the cells were incubated with GH alone during 30min-6h. When the cells were preincubated with GH during 3h, the lipolytic sensitivity for isoprenaline increased markedly without any change in maximal lipolysis. However, a full effect was only obtained if GH was also present during the incubation with isoprenaline. GH did not alter DB-CAMP, enprophylline, or forskolineinduced lipolysis in human fat cells. In conclusion, GH had no direct lipolytic effect on human fat cells but GH markedly increased the catecholamine sensitivity. The site of the GH effect seems to be in the i3-adrenoceptors or in the G, coupling protein.
Key Words: adipocytcs, lipolysis, glycerol, growth hormone, catccholaraincs It is well established that growth hormone (GH) is important not only for linear growth but also for adipocyte differentiation and energy metabolism in both animals and man. GH treatment of GH-deficient adults reduces the abdominal fat mass and decreases fat cell size (1,2). How these effects of GH on adipocyte cells are mediated is not yet fully understood. It seems to be established that GH reduces glucose uptake in the fat cells of humans (1,3) and rats (4), both basal and insulin-stimulated. GH weakly stimulates basal lipolysis in rat fat cells (5) and strongly stimulates theophyllamine and catecholamine lipolysis (6). In 3T3-F442A cells the lipolytic effect of GH seems to be due to a direct effect on the lipase (7). However, the acute effect of GH on lipolysis in human fat cells remains unclear. Incubation with GH did not affect lipolysis in human fat cells in culture (8) and GH treatment does not increase noradrenaline induced lipolysis in isolated abdominal and gluteal adipocytes from GH deficient children (1). In vivo, the lipolytic effect of GH in humans has been demonstrated both in adults and children using various methodological Adress correspondence to Dr Claude Marcus, Pediatric Endocrine Research Unit, Huddinge Hospital S-141 46 Huddinge, Sweden
1336
Lipolytic Effect of GH in Adipocytes
Vol. 54, No. 18, 1994
aproaches (9-11). This may indicate that the lipolytic effects of GH are mediated in different ways in humans than in other species. The aim of the present study was to reevaluate the effects of GH on basal lipolysis and lipolysis stimulated by both catecholamines and agents which stimulate lipolysis via activation of the lipolytic cascade at levels below the 6-adrenergic receptors in isolated adipocytes from healthy adults.
Material and methods
Abdominal subcutaneous adipose tissue was obtained during routine cholecystectomy and inguinal hernia operations on 17 non-obese patients, 12 males and 5 females, 18-40 years old. They were otherwise healthy, of normal weight and not on medication. After an overnight fast, general anesthesia was induced with thiopental sodium and maintained with a mixture of oxygen and nitrous oxide. The study was approved by the Ethics Committee of Karolinska Institute. Adipose tissue was removed from the surgical incision at the beginning of the operation. It was cut into fragments and isolated from the stroma by incubation with collagenase for 45 minutes in Krebs Ringer phosphate (KRP) buffer, pH 7.4, containing 4% bovine serum albumin (BSA), mainly using Rodbell's method (12). The adipocytes were washed in KRP albumin buffer and aggregated material was removed by filtration through a silk cloth. Lipolysis experiments. Fat cell suspensions (2%, vol/vol) were incubated in duplicate in KRP buffer (ph 7.4) with 40 mg/ml of BSA, 0.1 mg/ml of ascorbic acid and 1 mg/ml of glucose at 37°C for 30 min-6h. At the end of the incubations an aliquot of the medium was removed for determination of the glycerol release which was used as an index of lipolysis. In a subset of experiments, cells were preincubated with GH for 1-3h and then preincubated without GH for 0-2h as shown in Table 1. Thereafter and when the cells were between preincubations, they were washed twice with fresh KRP-albumin buffer and reincubated during the fourth hour with or without GH and isoprenaline in concentrations of 0, 10-13, 10-11 (submaximal concentrations) and 10 .8 mol/I (i.e., the isoprenaline concentration in our incubation system needed to obtain maximal lipolysis). In control experiments, the cells were treated in exactly the same way, but without GH. The glycerol concentration was analyzed by a sensitive kinetic bioluminescence method (13). Adenosine, secreted from fat cells, is a potent inhibitor of lipolysis. We have previously shown that the adenosine contamination is of no importance in this incubation system (14). All experiments were obtained on single adipose tissue samples. Chemicals. Recombinant GH was a gift from Kabi Pharmacia, Stockholm, Sweden. The collagenase was prepared from Clostridium histolyticum and was of Sigma type 1. Dialyzed BSA (fraction V) was purchased from the Armour Pharmaceutical Company, Eastbourne, England. The same batches of collagenase and albumin were used in all experiments. Isoprenaline, forskolin, enprophylline, dibutyryl cAMP and cycIoheximide came from Sigma Chemical Company, St. Louis, MO, USA. Statistics and analysis of results. The Student's paired and unpaired t-tests were used when appropriate.
Vol 54, No. 18, 1994
Lipolytic Effect of GH in Adipocytes
1337
Results Effects of GH on basal lipolysis. Adipocytes from three patients were incubated for 30min6h in the presence or absence of GH (7ng/ml). Lipolysis was constant for 6h - i.e., the glycerol concentration increased linearly (data not shown). No lipolytic or antilipolytic effect of GH was observed and the glycerol time-curves were identical, whether or not GH was present in the incubation medium.
Table
1
Effect of GH, during various incubation times, on lipolysis induced by submaximal isoprenaline concentrations Incubation hour 1 2 3 + + + +
+
+
+
+
+
+
lipolytic effect 0 0 (+) +
-
+
(+)
+
4 -
p
ns <0.05 ns
A d i p o c y t e s w e r e i n c u b a t e d d u r i n g four hours w i t h (+) or without (-) GH and, d u r i n g the f o u r t h hour, w i t h isoprenaline (I0-ii and 10-13 mol/l) as well. The l i p o l y t i c e f f e c t of i s o p r e n a l i n e alone, i° e., w i t h o u t GH, w h e r e the cells w e r e h a n d l e d the same way, was determined as control experiments, n=4, Lipolysis denotes the effect of GH compared to control e x p e r i m e n t s , ns = not s i g n i f i c a n t .
GH effect on isoprenaline-induced lipolysis. In adipocytes from four subjects, the effect on lipolysis of preincubation with GH (7ng/ml), induced by various concentrations of isoprenaline, was investigated, as described in Material and Methods and in Table 1. GH did not affect the maximal isoprenaline-induced lipolysis (Fig.l). However, the lipolytic effect of submaximal concentrations of isoprenaline was markedly enhanced by preincubation with GH for 3h. The effect of GH was significant only if GH was present in the incubation medium both during the whole preincubation period and during the incubation with isoprenaline (Table 1, Fig. 1). The effect of cycloheximide (protein synthesis inhibitor) on GH effets was investigated in seven patients. The effects of cycloheximide varied markedly between individuals. In three experiments cycloheximide markedly reduced the effect of GH on isoprenaline induced lipolysis without affecting the lipolysis induced by isoprenaline alone but in in three experiments no effect was observed and the overall effect of cycloheximide was not significant.
1338
Lipolytic Effect of GH in Adipocytes
Vol. 54, No. 18, 1994
A
Z0
100
B
VISO • ISO+GH • ISO+GH
/
i00 / '
75
////50 "J~""
50 H
25
'~////////
75
//I/ 25
q4 0 dO
0
-13
-ii Log
0
-8
Isoprenaline
-13
-ii
-8
Concentration
Fi.q. 1 The e f f e c t of GH on l i p o l y s i s i n d u c e d by i s o p r e n a l i n e . H u m a n a d i p o c y t e s w e r e p r e i n c u b a t e d for 3h w i t h (filled triangles) or w i t h o u t (open triangles) GH (7ng/ml) and thereafter washed and reincubated for lh with isoprenaline in the c o n c e n t r a t i o n s indicated (iso) alone (A,) or in c o m b i n a t i o n w i t h GH (B, filled triangles). * = p<0.05, Data p r e s e n t e d are m e a n s ± S D of four e x p e r i m e n t s m a d e in d u p l i c a t e .
Effect of GH on lipo!ysis induced by a.qents actin.q at postreceptor sites. In another set of experiments with adipocytes from four subjects, the lipolytic effects of enprophylline (phosphodiesterase inhibitor(15)), dibutyryI-CAMP (stable CAMP analogue) and forskolin (aclenylate cyclase activator (16)) were investigated either after preincubation with GH for 3h and with GH in the incubation medium or without GH. Adipocytes from the same subjects were also incubated with isoprenaline +GH, as previously described. Although that GH stimulated isoprenaline induced lipolysis significantly (p<0.05), the dose-response curves ±GH with agents stimulating lipolysis at a postreceptor level were identical (Fig.2).
Discussion In the present study we have shown that, in abdominal adipocytes from healthy adults, GH increases catecholamine lipolytic sensitivity without affecting the maximal effect induced by the drug. The effect of GH was seen after 3h preincubation, which is in agreement with previous in vivo results in humans (9,10) and with results from studies of
Vol. 54, No. 18, 1994
Lipolytic Effect of GH in Adipoeytes
1339
rat adipocytes (5,6). We were not able to demonstrate any direct lipolytic effect of GH in human adipocytes. This is in contrast to what has been reported in rat adipocytes and in 3T3 F442A, cells where GH has a direct stimulatory effect on lipolysis (5,7) and also increases the maximal effect of various agents such as theophylline and catecholamines (6,7). It has previously been reported that there is neither any lipolytic effect of GH in rabbits in vivo (17) nor in vitro (18). Our results are further indications of marked differences between species as regards the lipolytic effect of GH.
1.5
O ISO Q ISO+GH
1.5
O EHPRO • ENPRO+GH [~ FORSKOLIN m FORSKOLIN+GH
1.0
O o~ ~ ~0.5
0.5
0
-113
111
' -8
-'5 -'4
' -3
o
'
Log Drug Concentration,
-'9
-'8
"~
-'6
-'5
"4
'3
mol/l
The lipolytic effect of agents a c t i n g at p o s t r e c e p t o r sites with or w i t h o u t GH. Human adipocytes were p r e i n c u b a t e d for 3h w i t h GH and t h e r e a f t e r w a s h e d and reincubated for lh w i t h GH in combination with isoprenaline (iso), forskolin, dibutyryl-CAMP or e n p r o p h y l l i n e in the c o n c e n t r a t i o n s i n d i c a t e d (filled symbols). The cells were also preincubated and i n c u b a t e d w i t h o u t GH (open symbols) p r e s e n t in the i n c u b a t i o n medium.
In rat adipocytes the lipolytic effect of GH is depressed by cycloheximide which indicates that protein synthesis is involved (5). In the present study, the potentiating effect of GH on catecholamine induced lipolysis was partly depressed by cycloheximide only in some experiments. GH is secreted in pulses and it is possible that variations in the GH secretion pattern during the time period prior to the fat biopsy may explain the varying effect of cycloheximide. However, it is likely that GH influences lipolysis in more than one way as previously has been suggested (19). In weanling rats GH induces lipolysis without any lag period (20) and the same is observed in diabetic rats (21). Furthermore, in the present study, GH had to be present both during the whole preincubation period and the isoprenaline incubation period to produce the full effect, which also may indicate that GH
1340
Lipolytic Effect of GH in Adipocytes
Vol. 54, No. 18, 1994
has more effect on the lipolysis-promoting system than protein synthesis stimulation alone. GH failed to induce any increase in the lipolytic effect of forskolin, dibutyryl-CAMP or enprophyllin. All these agents activate lipolysis at a level below the 13-adrenergic receptors and the G s protein. Thus, it is likely that the effect of GH is due to changes in 13adrenoceptor number or functional coupling to the G s protein. This is well in accordance with the observation that GH increases sensitivity but not the maximal response of isoprenaline. It has previously been shown that there are spare g-adrenoceptors on human fat cells (22). Thus, an increased number of functionally coupled receptors, would only increase the sensitivity.
Acknowled,qements This study was supported by grants from the Swedish Society of Medicine, the Swedish Medical Research Council (19X-9941), Karolinska Institute, Harald and Greta Jeansson, the Golje Memorial, The Crown Princess Lovisa/Thileman, the Swedish Diabetes, the General Maternity Hospital, the Nordic Insulin and the Eriksson Research Foundations.
References 1. M. ROSENBAUM, J. GERTNER and R. LEIBEL. J Clin Endocrinol Metab. 6._9_912741281 (1989). 2. R. J. MARTIN, M. DREWRY, D. JEWELL, R. HARRIS, R. YOUNG and J. S. PATTON. Int J Obesity 1.__33327-335, (1989). 3. G. NYBERG, S. BOSTROM, R. JOHANSSON and U SMITH. Acta Endocrinologica 9_.55 129-133, (1980). 4. B. MALOFF, J. LEVINE and D. LOCKWOOD. Endocrinology 107:538-544 (1980). 5. A.B. CALDWELL and J.N. FAIN. Horm Metab Res 23-5, (1970). 6. M GOODMAN. Endocrinology 114131-135, (1984). 7. J. DIETZ and J. SCHWARTZ. Metabolism, 40800-806, (1991). 8. G. NYBERG and U. SMITH. Horm Metab Res 9:22-27, (1977). 9. P. METCALFE, D.G. JOHNSTON, R. NOSADINI, H. 0RSKOV, K. G. ALBERTI. Diabetologia 2__0123-128, (1981). 10. G. VAN VLIET, D. BOSSON, M. CRAEN, M. DU CAJU, P. MALVAUX and M. VANDERSCHUEREN-LODEWEYCKS. J Clin Endocrinol Metab 6._.55876-879 (1987). 11. P. BOYLE, A. AVOGARO, L. SMITH, D. BIER, A. PAPPU, R. ILLINGWORTH AND P. CRYER. Am J. Physiol. 263 E168-E172, (1992). 12. M. RODBELL. J. Biol. Chem. 239375-80 (1964). 13. J. HELLMER, P. ARNER AND A LUNDIN Analythical Biochem 177132-37 (1989). 14. C. MARCUS, B. KARPE, P. BOLME, T. SONNENFELD AND P. ARNER. J. Clin. Invest. 7.__991812-18(1987). 15. B. FREDHOLM and E. LINDGREN Acta Pharmacol. Toxicol. 5.~46469 (1983). 16. K. SEAMON, W. PADGETT, and J. DALY. Proc. Natl. Acad. Sci. 7._883363-3367, (1981). 17. J. KNUDTZON, P.D. EDMINSON, K.L. REICHELT. Hormone Res 2110-18, (1985). 18. C. BOWDEN, K. WHITE, U. LEWIS and G. TUTWEILER. Metabolism. 4237-243, (1985).
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19. H. M. GOODMAN. Endocrinology, 109, 120-129, (1981). 20. H.M. GOODMAN, V. CORIO. Endocrinology 109 2046-2053, (1981). 21. S. SOLOMON, S. D. SIBLEY, T. M. CUNNINGHAM. Endocrinology 127., 1544-46, (1990). 22. P. ARNER, J. HELLMER, A. WENNLUND, J. OSTMAN and P. ENGFELDT. Eur J Pharm 146 45-56 (1983).