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Effect of amitriptyline on lipolysis and cyclic AMP concentration in isolated fat cells
Effect of Amitriptyline on Lipolysis and Cyclic AMP Concentration in Isolated Fat Cells By Fred C. Lovrien,
Ann A. Steele,
Joseph
The effect of amitriptyline on lipolysis and intracellular concentration of cyclic AMP in vitro was examined in fat cells isolated from epididymal adipose tissue of fasted rats. Incubations were performed in the absence of glucose. Amitriptyline increased the intracellular concentration of cyclic AMP in isolated fat cells. It also markedly enhanced the intracellular concentration of cyclic AMP caused by DLarterenol plus theophylline. Despite increasing the intracellular concentration of cyclic AMP amitriptyline did not
D. Brown,
and Daniel
B. Stone
stimulate basal lipolysis and it markedly inhibited lipolysis caused by DL-arterenol and dibutyryl cyclic AMP plus theophylline. The mechanism by which amitriptyline is antilipolytic and yet increases the concentration of cyclic AMP is unknown. It is possible that amitriptyline inhibits the binding of cyclic AMP to phosphodiesterase and also inhibits the binding of cyclic AMP to a protein kinase or triglyceride lipase, or that amitriptyline directly inhibits triglyceride lipase.
ARIOUS AGENTS, including DL-arterenol, theophylline, and dibutyryl cyclic AMP, have been found to accelerate the release of fatty acids and glycerol in vitro from isolated fat cells of the rat. l-3 These lipolytic effects are inhibited by insulin, I-butyl-3-tolysulfonylurea (tolbutamide), and phenethylbiguanide (phenformin). 4-7 The inhibition of lipolysis caused by insulin is associated with a decrease in the concentration of cyclic AMP in the isolated fat cells The present experiments were designed to observe the effects of amitriptyline (Elavil) on lipolysis and the intracellular concentration of cyclic AMP in isolated fat cells prepared from the epididymal fat pads of fasted rats. Lipolysis was stimulated by DL-arterenol, theophylline, and dibutyryl cyclic AMP plus theophylline. The results indicate that amitriptyline is a potent inhibitor of lipolysis in isolated fat cells but in contrast to insulin causes an increase in the intracellular concentration of cyclic AMP. MATERIALS
AND
METHODS
Male Wistar rats (1.40-180 g) that had been fed a high-fat diet (40%) for 7-10 days were deprived of food for 24 hr before the experiments and were killed by decapitation after stunning. Isolated fat cells were prepared by Rodbell’s technique9 in which pieces of epididymal adipose tissue were incubated with crude collagenase for I hr in the albumin bicarbonate buffer solution described by Fain. 10 Glucose was omitted from the medium during treatment of the adipose tissue with collagenase, during subsequent washing of the
From the Department of Internal Medicine, Uniz)ersify Hospifals, Iowa City, Iowa. Received for publication July 22, 1971. Supported by USPHS Research Grant AM-11465, National Institute of Arthritis and Metabolic Diseases, and Iowa Hearf Association Grant 70-G-24 GP540. Fred C. Lovrien, B.A.: 7unior Medical Student, University of Iowa College of Medicine, Iowa City, lowa. Ann A. Steele, M.D.: Doctor’s Clinic, Bremerton, Wash. Joseph D. Brown, M.D.: Associate Professor, Department of Internal Medicine, University Hospitals, Iowa City, Iowa. Daniel B. Stone, M.D.: Professor and Vice-Chairman, Department of Medicine, University of Nebraska, Omaha, Nebr.
Metabolism, Vol. 21, No. 3 (March), 1972
223
224
LOVRIEN
ET AL.
cells, and during incubation. A I% solution of albumin (Bovine Fraction V, Armour) in bicarbonate buffer was prepared daily; the pH was adjusted to 7.4 in an atmosphere of 5% CO, and 95% 0,. This buffer solution was filtered through a millipore filter (pore size 0.4 CL)before use. Each day the fat cells from two rats were pooled and distributed equally among a number of flasks. Incubation was performed in groups of six flasks in each daily experiment. Initial control values were obtained after fat cells had been incubated for five minutes. The quantity of fat cells in each flask was determined by analysis of the total fatty acid content of the flask. One gram of adipose tissue has been reported to contain approximately 3mM of fatty acid,11 and this concentration of fatty acids in adipose tissue was confirmed in our laboratory using both adipose tissue before and after incubation with collagenase. Aliquots of the medium were analyzed for glycerol concentration by the method of Weiland and Suyterl2313 and for free fatty acids (FFA) by a modification of the method of Dole and Meinertz.14115 The concentration of cyclic AMP in isolated fat cells was measured by the method of Goldberg.16 After preparing the free fat cells and washing them to remove the collagenase, the cells were distributed into polyethylene flasks containing the appropriate reagents and the albumin bicarbonate buffer. The cells were incubated for eight minutes. The contents of the six flasks in each group were then pooled and the isolated fat cells immediately removed from the buffer by centrifugation. The cells were extracted with 10% trichloroacetic acid (TCA) and then analyzed for cyclic AMP content. Selected incubation flasks were analyzed for total fatty acid content. The total fatty acid content of six pooled flasks was approximately 350 pmoles of triglyceride representing about 350 mg of adipose tissue. DL-arterenol hydrochloride was purchased from the Sigma Chemical Company and dibutyryl cyclic AMP from Cal Biochem. Theophylline was purchased locally. Amitriptyline was supplied by Merck, Sharp and Dohme. The collected data from the experiments were analyzed for variance. Treatment means were compared using Tukey’s test.17
RESULTS
DL-arterenol, dibutyryl cyclic AMP, and theophylline were used to accelerate the release of free fatty acids and glycerol by isolated fat cells. Figure I shows the dose response curve of the antilipolytic effect of amitriptyline when lipolysis was stimulated by DL-arterenol (1 pg/ml). In the absence of amitriptyline the release of free fatty acids caused by DL-arterenol was 24 rmoles/g of adipose tissue (mean of six flasks.) Each point on the dose response curve was the mean of the results from six incubation flasks. It can be seen that the steep portion of the dose response lies between a concentration of 1 and 10 mg/lOO ml of amitriptyline. Further studies with amitriptyline were done at a concentration of 5 mg/lOO ml. Figure 2 shows the dose response curve of the antilipolytic effects of amitriptyline when lipolysis was stimulated by dibutyryl cyclic AMP (1 mM) plus theophylline (10m4 M). Again it can be seen that the steep portion of the dose response curve lies between a concentration of 1 and 10 mg/lOO ml of amitriptyline. Table 1 lists the effect of amitriptyline (5 mg/lOO ml) on basal lipolysis and lipolysis stimulated with DL-arterenol (1 pglml), dibutyryl cyclic AMP (1 mM) plus theophylline (10 -* M), and theophylline alone (low3 M). Amitriplyline had no effect on basal lipolysis. It can be seen in the table, however, that amitriptIyIine was a potent inhibitor of 1ipoIysis induced by DL-arterenol, dibutyryl cyclic AMP plus theophylline, and theophylline alone. Table 2 shows the effect of theophylline, amitriptyline, DL-arterenol plus theophylline and
225
AMITRIPTYLINE
. 2o-
\ * -2
’ -I
;
I
Fig. 1. Effect of varying concentrations of amitriptyline on DL-arterenol (1 Wg/ml) induced lipolysis in isolated fat cells. In absence of amitriptyline, free fatty acid release induced by DL-arterenol was 24 umoleslg.
:N 2
LOG,, CONC. AYITRIPTYLINEknqa/loo ml.1
plus theophylline plus amitriptyline on the concentration of AMP and glycerol release in isolated fat cells. The cells were incubated for 8 min. Basal cyclic AMP concentrations in isolated fat cells are very low. For this reason all incubations were carried out in the presence of either theophylline or amitriptyline to act as a phosphodiesterase inhibitor. Amitriptyline produced no significant change in cyclic AMP concentration or glycerol release when compared to theophylline alone. It can be seen, however, that DL-arterenol plus theophylline not only induced a significant increase in the intracellular concentration of cyclic AMP but also caused a significant increase in glycerol release when compared with theophylline or amitriptyline alone. When the isolated cells were incubated with DL-arterenol plus theophylline plus amitriptyline (5 mg/100 ml) there was a further increase in the concentration of cyclic AMP but no significant change in glycerol release compared to DL-arterenol plus theophylline. DL-arterenol
cyclic
Table 1. Effect of Amitriptyline
on Basal and Induced FFA Release knot&g)
Conditions
Basal Basal + amitriptyline Basal DL-arterenol DL-arterenol Basal
(1 Bg/ml) (1 pg/ml)
(5 mg/iOO
+ amitriptyline
(IO-” M) + amitriptyline
Glycerol Release (fimoleslg)
5+1 6?1
ml)
Dibutyryl C-AMP (1 mM) + theophylline Dibutyryl C-AMP (1 mM) + theophylline amitriptyline (5 mg/lOO ml) Basal Theophylline (10“ M) Theophylline
Lipolysis”
(5 mg/lOO (lo-’ (10“
ml)
M) M) +
(5 mg/lOO
ml)
5+2 83 2 28 + 21’1 92 + 45 r 3*1 60 f 27 f
10 t 1 4+1 422 2t 2*
68 2 2t 25 + 2* 621
1t 1*
58 2 lt 31 r 1*
lt l$
4 2 0.5 26 2 0.5t 9 +- 0.5$
*Free fat Cells (4-10 mgiflask) were incubated for 1 hr in 2 ml of glucose-free Values are mean * SE Of at least five daily experiments. SE is based on pooled of variance from analysis of variance. tP < 0.05 for difference between basal and induced lipolysis. *P < 0.05
for difference of amitriptyline
plus lipolytic
agent from
lipolytic
medium. estimate
agent
alone.
226
LOVRIEN
Fig. 2. Effect of varying concentrations of amitriptyline on dibutyryl cyclic AMP (1mM) plus theophylline (1O-4 M) induced lipolysis in isolated fat cells. In absence of amitriptyline, free fatty acid release induced by dibutyryl cyclic AMP plus theophylline was 70 Wmoles/g.
Lo6mCONC. AWITRIPTYLINE
ET AL.
(nqs1100ml)
DISCUSSION Recent investigators have clarified the effects of various agents on the lipolytic enzyme system of adipose tissue. It has been suggested that catecholamines interact with a beta adrenergic receptor causing the formation, via adenyl cyclase, of cyclic AMP which in turn activates triglyceride lipase. Recent evidence suggests that cyclic AMP does not directly activate triglyceride lipase but instead stimulates a protein kinase18 which is then involved in the activation of triglyceride lipase. Amitriptyline and other tricyclic antidepressants have recently been shown in vitro to be potent inhibitors of the cyclic 3’, SAMP phosphodiesterase obtained from heart muscle.‘g*20 It has been reported that patients with depressive illnesses have decreased urinary excretion of cyclic AMI’lgv21 and that treatment with the tricyclic antidepressants, including amitriptyline, increases Table 2. Effect
of Amitriptyline and Various Lipolytic Agents on Cyclic and Glycerol Release in Isolated Fat Cells* (10-l nmoles/g) C-AMP Concentration
Conditions
Theophylline (10m4M) Amitriptyline (5 mg/lOO ml) DL-arterenol (1 ug/ml) + theophylline DL-arterenol (1 ag/ml) + theophylline + amitriptyline (5 mg/lOO ml) *Free fat cells (approximately 60 free medium. Values are mean (* estimate of variance from analysis tween effects of theophylline and glycerol re!ease with DL-arterenol line.
12.02 + 2.77 (10m4M) (lo-’ M)
AMP Concentration
Glycerol Release (Etmoleslg)
0.24 0.12 2.30 1.68
10.29 + 2.77 58.33 + 2.77T 89.47 f 2.77$
+ -c f 2
0.10 0.10 O.lOT 0.10
mg/flask) were incubated for 8 min in 2 ml of glucoseSE) of six daily experiments. SE is based on pooled of variance. There was no significant difference beamitritpyline and no significant difference between plus theophylline in presence or absence of amitripty-
Tp < 0.05 for effect of DL-arterenol amitriptyline alone. Sp < 0.05 for effect of DL-arterenol DL-arterenol plus theophylline alone.
plus
theophylline
plus theophylline
compared plus
to theophylline
amitriptyline
compared
or to
AMITRIPTYLINE
227
the urinary excretion of cyclic AMP. This increase in urinary cyclic AMP excretion may be directly related to therapy with the antidepressants, but it may also be related to other metabolic changes or to increased exercise as the depressive illness improves. Theophylline, a potent inhibitor of phosphodiesterase, is known to stimulate lipolysis in vitro in isolated fat cells. Theophylline also increases the intracellular concentration of cyclic AMP.* Presumably the increase in lipolysis is secondary to the increased concentrations of intracellular cyclic AMP. Amitriptyline inhibits cyclic 3’,5’AMP phosphodiesterase in heart muscle and may have similar effects on adipose tissue. As shown in Table 2, amitriptyline, like theophylline, increased the concentration of intraceullar cyclic AMP. It also markedly increased the concentration of cyclic AMP caused by DL-arterenol plus theophylline. These results are consistent with the interpretation that amitriptyline is an inhibitor of phosphodiesterase. In contrast to theophylline, however, the data in Table 1 indicate that amitriptyline does not stimulate basal lipolysis and is in fact a potent inhibitor of induced lipolysis in spite of its ability to enhance markedly the intracellular concentration of cyclic AMP. The data given in Table 2 were obtained when the isolated fat cells were incubated for 8 min. It has been demonstrated that the maximal concentration of cyclic AMP in isolated fat cells occurs within approximately 6-8 min and slowly declines with 15-20 min of incubation.* The data of Butcher et al. suggests that cyclic 3’SAMP is responsible for activating lipolysis, but the rate of lipolysis does not parallel cyclic AMP concentration beyond approximately a twofold increase over basal levels. The methyl xanthines have provided amplification of the cyclic AMP response to hormones that stimulate adenyl cyclase, thus facilitating studies not only of these hormones but also of agents that act to decrease cyclic AMP levels. It can be seen in Table 2 that after 8 min of incubation with DL-arterenol plus theophylline, there was a marked increase in the concentration of cyclic AMP and also a significant increase in the release of glycerol. Amitriptyline in the presence of DL-arterenol and theophylline gave a further increase in the concentration of cyclic AMP but no change in glycerol release. If the incubations are continued for 1 hr, however, amitriptyline, as shown in Table 1, is a potent inhibitor of lipolysis induced by DL-arterenol plus theophylline, despite the fact that it enhances the initial concentration of cyclic AMP in the fat cell. It has recently been demonstrated that both deoxyfrenolicin and Vitamin K, are antilipolytic but act synergistically with norepinephrine or corticotropin to elevate intracellular levels of cyclic AMP in isolated fat cells.22.23 It has been suggested that cyclic AMP in adipose tissue cells may be present in different compartments and all of the cyclic AMP may not be available for stimulating lipolysis. 24,25 The concentration of cyclic AMP required for the activation of lipase may be very critical and excess cyclic AMP may cause a reduction in lipase activity.26. The mechanism by which amitriptyline inhibits lipolysis and yet increases the intracellular concentration of cyclic AMP is unknown. The fact that amitriptyline can inhibit lipolysis under conditions where cyclic AMP concentration is increased suggests that the antilipolytic effect of amitriptyline
LOVRIEN
228
ETAL.
results from the inhibition of the lipolytic enzyme system that is activated by cyclic AMP, rather than the effect of amitriptyline on cyclic AMP concentration itself. The increase in the intracellular concentration of cyclic AMP caused by amitriptyline may be due to the inhibition of phosphodiesterase. It is possible that amitriptyline inhibits the binding of cyclic AMP not only to phosphodiesterase but also inhibits cyclic AMP binding to a protein kinase or triglyceride lipase. This would inhibit lipolysis by blocking the activation of triglyceride lipase and at the same time there would be an increase in intracellular concentration of cyclic AMP because of the inability of phosphodiesterase to convert the cyclic AMP to SAMP. It is also possible that amitriptyline directly inhibits triglyceride lipase. ACKNOWLEDGMENT We are grateful to Dr. Nelson Goldberg and Dr. Joseph Larner for their help in teaching us to assay cyclic AMP and helping in the preparation of phosphodiesterase. REFERENCES 1. Hollenburg, C. H.: Adipose tissue lipases. II. In Renold, A. E., and Cahill, G. F. (Eds.): Handbook of Physiology, Section 5: Adipose Tissue. Washington, D. C., American Physiology Society, 1965. 2. Rodbell, M.: The metabolism of isolated fat cells. In Renold, A. E., and Cahill, G. F. (Eds.): Handbook of Physiology, Section 5: Adipose Tissue. Washington, D. C., American Physiology Society, 1965. 3. Butcher, R. W., Ho, R. J., Meng, H. D., and Sutherland, E. W.: Adenosine 3’, 5’monophosphate in biological materials. II. The measurement of adenosine 3’, 5’-monophosphate in tissues and the role of the cyclic nucleotide in the lipolytic response of fat to epinephrine. J. Biol. Chem. 240: 4515,1965. 4.
Fain, J. N., Kovacev, V. M., and Scow, R. 0.: Antilipolytic effect of insulin in isolated fat cells of the rat. Endocrinology 78: 773, 1966. 5. Brown, J. D., and Stone, D. B.: Antilipolytic effects of sulfonylurea drugs on induced lipolysis in isolated fat cells of the rat. Endrocrinology 81:71, 1967. 6. Stone, D. B., and Brown, J. D.: In vitro effects of phenformin hydrochloride: Observations using isolated fat cells. Ann. N. Y. Acad. Sci. 148:623, 1968. 7. Brown, J. D., Stone, D. B., and Steele, A. A.: The mechanism of action of antilipolytic agents: A comparison of the effects of insulin, tolbutamide, and phenformin on lipolysis induced by dibutyryl cyclic AMP plus theophylline. Metabolism 18:926, 1969.
8. Butcher, R. W., Baird, C. E., and Suthand erland, E. W.: Effects of lipolytic antilipolytic substances on adenosine 3’, s’monophosphate levels in isolated fat cells. J. Biol. Chem. 243:1705, 1968. 9. Rodbell, M.: Metabolism of isolated fat cells. I. Effects of hormones on glucose metabolism and lipolysis. J. Biol. Chem. 239:375,1964. 10. Fain, J. N.: Effect of dexamethasone and growth hormone on fatty acid mobilization and glucose utilization in adrenalectomized rats. Endrocrinology 71:633, 1962. II. Rodbell, M.: Modulation of lipolysis in adipose tissue by fatty acid concentration in fat cell. Ann. N. Y. Acad. Sci. 131: 302, 1965.
0.: Eine Enzymatische 12. Wieland, Methode zur Bestimmung von Glycerin. Biothem.
Z. 329:313, 1957.
13. -,
and Suyter, M.: Glycerokinase: Isolierung und Eigenschaften des Enzyms. Biochem. Z. 329:330,1957. 14. Stone, D. B., Brown, J. D., and Cox, C. P.: Effect of tolbutamide and phenformin on lipolysis in adipose tissue in vitro. Amer. J. Physiol. 210:26, 1966. 15. Dole, V. I’., and Meinertz, H.: Microdetermination of long-chain fatty acid in plasma and tissues. J. Biol. Chem. 235: 2595, 1960. 16. Goldberg, N. D., Villar-Palasi, C., Sasko, H., and Larner, J.: Effects of insulin treatment on muscle 3’, 5’-cyclic adenylate levels in vivo and in vitro. Biochem. Biophys. Acta 148:665, 1967.
229
AMITRIPTYLINE
17. Huntsberger, D. V., and Leaverton, P. E.: Statistical inference in the biomedical sciences. Boston, Allyn & Bacon, 1970, p. 211. 18. Corbin, J. D., and Krebs, E. G.: A cyclic AMP stimulated protein kinase in adipose tissue. Biochem. Biophys. Res. Commun. X:328, 1969. 19. Abdulla, Y. H., and Hamadah, K.: 3’, 5’-cyclic adenosine monophosphate in depression and mania. Lancet 1:378, 1970. 20. Ramsden, N.: Cyclic AMP in depression and mania. Lancet 2 :lOS,1970. 21. Paul, M. I., Kitzion, B. R., and Janowsky, D. S.: Affective illness and cyclic AMP excretion. Lancet 1:88, 1970. 22. Kuo, J. F.: Effects of deoxyfrenolicin on isolated adipose cells. II. Lipolysis adenosine 3’, 5’-monophosphate levels, and comparison with the effects of Vitamin K,. Biothem. Pharmacol. 18:757, 1969.
23. -, and Greengard, I’.: Cyclic nucleotide-dependent protein kinases. J. Biol. Chem. 243~4067, 1970. 24. Butcher, R. W., Sneyd, J. G. T., Park, C. R., and Sutherland, E. W., Jr.: Effect of insulin on adenosine 3’, 5’-monophosphate in the rat epididymal fat pad. J. Biol. Chem. 241:1651, 1966. 25. Kuo, J. F., and DeRenzo, E. C.: A comparison of the effects of lipolytic and antilipolytic agents on adenosine 3’, Smonophosphate levels in adipose cells as determined by prior labeling with adenineB-14C. J. Biol. Chem. 244 :2252,1969. 26. Rizack, M. A.: Activation of an epinephrine-sensitive lipolytic activity from adipose tissue by adenosine 3’, $-phosphate. J. Biol. Chem. 239:392, 1964.
Ann A. Steele,
Joseph
The effect of amitriptyline on lipolysis and intracellular concentration of cyclic AMP in vitro was examined in fat cells isolated from epididymal adipose tissue of fasted rats. Incubations were performed in the absence of glucose. Amitriptyline increased the intracellular concentration of cyclic AMP in isolated fat cells. It also markedly enhanced the intracellular concentration of cyclic AMP caused by DLarterenol plus theophylline. Despite increasing the intracellular concentration of cyclic AMP amitriptyline did not
D. Brown,
and Daniel
B. Stone
stimulate basal lipolysis and it markedly inhibited lipolysis caused by DL-arterenol and dibutyryl cyclic AMP plus theophylline. The mechanism by which amitriptyline is antilipolytic and yet increases the concentration of cyclic AMP is unknown. It is possible that amitriptyline inhibits the binding of cyclic AMP to phosphodiesterase and also inhibits the binding of cyclic AMP to a protein kinase or triglyceride lipase, or that amitriptyline directly inhibits triglyceride lipase.
ARIOUS AGENTS, including DL-arterenol, theophylline, and dibutyryl cyclic AMP, have been found to accelerate the release of fatty acids and glycerol in vitro from isolated fat cells of the rat. l-3 These lipolytic effects are inhibited by insulin, I-butyl-3-tolysulfonylurea (tolbutamide), and phenethylbiguanide (phenformin). 4-7 The inhibition of lipolysis caused by insulin is associated with a decrease in the concentration of cyclic AMP in the isolated fat cells The present experiments were designed to observe the effects of amitriptyline (Elavil) on lipolysis and the intracellular concentration of cyclic AMP in isolated fat cells prepared from the epididymal fat pads of fasted rats. Lipolysis was stimulated by DL-arterenol, theophylline, and dibutyryl cyclic AMP plus theophylline. The results indicate that amitriptyline is a potent inhibitor of lipolysis in isolated fat cells but in contrast to insulin causes an increase in the intracellular concentration of cyclic AMP. MATERIALS
AND
METHODS
Male Wistar rats (1.40-180 g) that had been fed a high-fat diet (40%) for 7-10 days were deprived of food for 24 hr before the experiments and were killed by decapitation after stunning. Isolated fat cells were prepared by Rodbell’s technique9 in which pieces of epididymal adipose tissue were incubated with crude collagenase for I hr in the albumin bicarbonate buffer solution described by Fain. 10 Glucose was omitted from the medium during treatment of the adipose tissue with collagenase, during subsequent washing of the
From the Department of Internal Medicine, Uniz)ersify Hospifals, Iowa City, Iowa. Received for publication July 22, 1971. Supported by USPHS Research Grant AM-11465, National Institute of Arthritis and Metabolic Diseases, and Iowa Hearf Association Grant 70-G-24 GP540. Fred C. Lovrien, B.A.: 7unior Medical Student, University of Iowa College of Medicine, Iowa City, lowa. Ann A. Steele, M.D.: Doctor’s Clinic, Bremerton, Wash. Joseph D. Brown, M.D.: Associate Professor, Department of Internal Medicine, University Hospitals, Iowa City, Iowa. Daniel B. Stone, M.D.: Professor and Vice-Chairman, Department of Medicine, University of Nebraska, Omaha, Nebr.
Metabolism, Vol. 21, No. 3 (March), 1972
223
224
LOVRIEN
ET AL.
cells, and during incubation. A I% solution of albumin (Bovine Fraction V, Armour) in bicarbonate buffer was prepared daily; the pH was adjusted to 7.4 in an atmosphere of 5% CO, and 95% 0,. This buffer solution was filtered through a millipore filter (pore size 0.4 CL)before use. Each day the fat cells from two rats were pooled and distributed equally among a number of flasks. Incubation was performed in groups of six flasks in each daily experiment. Initial control values were obtained after fat cells had been incubated for five minutes. The quantity of fat cells in each flask was determined by analysis of the total fatty acid content of the flask. One gram of adipose tissue has been reported to contain approximately 3mM of fatty acid,11 and this concentration of fatty acids in adipose tissue was confirmed in our laboratory using both adipose tissue before and after incubation with collagenase. Aliquots of the medium were analyzed for glycerol concentration by the method of Weiland and Suyterl2313 and for free fatty acids (FFA) by a modification of the method of Dole and Meinertz.14115 The concentration of cyclic AMP in isolated fat cells was measured by the method of Goldberg.16 After preparing the free fat cells and washing them to remove the collagenase, the cells were distributed into polyethylene flasks containing the appropriate reagents and the albumin bicarbonate buffer. The cells were incubated for eight minutes. The contents of the six flasks in each group were then pooled and the isolated fat cells immediately removed from the buffer by centrifugation. The cells were extracted with 10% trichloroacetic acid (TCA) and then analyzed for cyclic AMP content. Selected incubation flasks were analyzed for total fatty acid content. The total fatty acid content of six pooled flasks was approximately 350 pmoles of triglyceride representing about 350 mg of adipose tissue. DL-arterenol hydrochloride was purchased from the Sigma Chemical Company and dibutyryl cyclic AMP from Cal Biochem. Theophylline was purchased locally. Amitriptyline was supplied by Merck, Sharp and Dohme. The collected data from the experiments were analyzed for variance. Treatment means were compared using Tukey’s test.17
RESULTS
DL-arterenol, dibutyryl cyclic AMP, and theophylline were used to accelerate the release of free fatty acids and glycerol by isolated fat cells. Figure I shows the dose response curve of the antilipolytic effect of amitriptyline when lipolysis was stimulated by DL-arterenol (1 pg/ml). In the absence of amitriptyline the release of free fatty acids caused by DL-arterenol was 24 rmoles/g of adipose tissue (mean of six flasks.) Each point on the dose response curve was the mean of the results from six incubation flasks. It can be seen that the steep portion of the dose response lies between a concentration of 1 and 10 mg/lOO ml of amitriptyline. Further studies with amitriptyline were done at a concentration of 5 mg/lOO ml. Figure 2 shows the dose response curve of the antilipolytic effects of amitriptyline when lipolysis was stimulated by dibutyryl cyclic AMP (1 mM) plus theophylline (10m4 M). Again it can be seen that the steep portion of the dose response curve lies between a concentration of 1 and 10 mg/lOO ml of amitriptyline. Table 1 lists the effect of amitriptyline (5 mg/lOO ml) on basal lipolysis and lipolysis stimulated with DL-arterenol (1 pglml), dibutyryl cyclic AMP (1 mM) plus theophylline (10 -* M), and theophylline alone (low3 M). Amitriplyline had no effect on basal lipolysis. It can be seen in the table, however, that amitriptIyIine was a potent inhibitor of 1ipoIysis induced by DL-arterenol, dibutyryl cyclic AMP plus theophylline, and theophylline alone. Table 2 shows the effect of theophylline, amitriptyline, DL-arterenol plus theophylline and
225
AMITRIPTYLINE
. 2o-
\ * -2
’ -I
;
I
Fig. 1. Effect of varying concentrations of amitriptyline on DL-arterenol (1 Wg/ml) induced lipolysis in isolated fat cells. In absence of amitriptyline, free fatty acid release induced by DL-arterenol was 24 umoleslg.
:N 2
LOG,, CONC. AYITRIPTYLINEknqa/loo ml.1
plus theophylline plus amitriptyline on the concentration of AMP and glycerol release in isolated fat cells. The cells were incubated for 8 min. Basal cyclic AMP concentrations in isolated fat cells are very low. For this reason all incubations were carried out in the presence of either theophylline or amitriptyline to act as a phosphodiesterase inhibitor. Amitriptyline produced no significant change in cyclic AMP concentration or glycerol release when compared to theophylline alone. It can be seen, however, that DL-arterenol plus theophylline not only induced a significant increase in the intracellular concentration of cyclic AMP but also caused a significant increase in glycerol release when compared with theophylline or amitriptyline alone. When the isolated cells were incubated with DL-arterenol plus theophylline plus amitriptyline (5 mg/100 ml) there was a further increase in the concentration of cyclic AMP but no significant change in glycerol release compared to DL-arterenol plus theophylline. DL-arterenol
cyclic
Table 1. Effect of Amitriptyline
on Basal and Induced FFA Release knot&g)
Conditions
Basal Basal + amitriptyline Basal DL-arterenol DL-arterenol Basal
(1 Bg/ml) (1 pg/ml)
(5 mg/iOO
+ amitriptyline
(IO-” M) + amitriptyline
Glycerol Release (fimoleslg)
5+1 6?1
ml)
Dibutyryl C-AMP (1 mM) + theophylline Dibutyryl C-AMP (1 mM) + theophylline amitriptyline (5 mg/lOO ml) Basal Theophylline (10“ M) Theophylline
Lipolysis”
(5 mg/lOO (lo-’ (10“
ml)
M) M) +
(5 mg/lOO
ml)
5+2 83 2 28 + 21’1 92 + 45 r 3*1 60 f 27 f
10 t 1 4+1 422 2t 2*
68 2 2t 25 + 2* 621
1t 1*
58 2 lt 31 r 1*
lt l$
4 2 0.5 26 2 0.5t 9 +- 0.5$
*Free fat Cells (4-10 mgiflask) were incubated for 1 hr in 2 ml of glucose-free Values are mean * SE Of at least five daily experiments. SE is based on pooled of variance from analysis of variance. tP < 0.05 for difference between basal and induced lipolysis. *P < 0.05
for difference of amitriptyline
plus lipolytic
agent from
lipolytic
medium. estimate
agent
alone.
226
LOVRIEN
Fig. 2. Effect of varying concentrations of amitriptyline on dibutyryl cyclic AMP (1mM) plus theophylline (1O-4 M) induced lipolysis in isolated fat cells. In absence of amitriptyline, free fatty acid release induced by dibutyryl cyclic AMP plus theophylline was 70 Wmoles/g.
Lo6mCONC. AWITRIPTYLINE
ET AL.
(nqs1100ml)
DISCUSSION Recent investigators have clarified the effects of various agents on the lipolytic enzyme system of adipose tissue. It has been suggested that catecholamines interact with a beta adrenergic receptor causing the formation, via adenyl cyclase, of cyclic AMP which in turn activates triglyceride lipase. Recent evidence suggests that cyclic AMP does not directly activate triglyceride lipase but instead stimulates a protein kinase18 which is then involved in the activation of triglyceride lipase. Amitriptyline and other tricyclic antidepressants have recently been shown in vitro to be potent inhibitors of the cyclic 3’, SAMP phosphodiesterase obtained from heart muscle.‘g*20 It has been reported that patients with depressive illnesses have decreased urinary excretion of cyclic AMI’lgv21 and that treatment with the tricyclic antidepressants, including amitriptyline, increases Table 2. Effect
of Amitriptyline and Various Lipolytic Agents on Cyclic and Glycerol Release in Isolated Fat Cells* (10-l nmoles/g) C-AMP Concentration
Conditions
Theophylline (10m4M) Amitriptyline (5 mg/lOO ml) DL-arterenol (1 ug/ml) + theophylline DL-arterenol (1 ag/ml) + theophylline + amitriptyline (5 mg/lOO ml) *Free fat cells (approximately 60 free medium. Values are mean (* estimate of variance from analysis tween effects of theophylline and glycerol re!ease with DL-arterenol line.
12.02 + 2.77 (10m4M) (lo-’ M)
AMP Concentration
Glycerol Release (Etmoleslg)
0.24 0.12 2.30 1.68
10.29 + 2.77 58.33 + 2.77T 89.47 f 2.77$
+ -c f 2
0.10 0.10 O.lOT 0.10
mg/flask) were incubated for 8 min in 2 ml of glucoseSE) of six daily experiments. SE is based on pooled of variance. There was no significant difference beamitritpyline and no significant difference between plus theophylline in presence or absence of amitripty-
Tp < 0.05 for effect of DL-arterenol amitriptyline alone. Sp < 0.05 for effect of DL-arterenol DL-arterenol plus theophylline alone.
plus
theophylline
plus theophylline
compared plus
to theophylline
amitriptyline
compared
or to
AMITRIPTYLINE
227
the urinary excretion of cyclic AMP. This increase in urinary cyclic AMP excretion may be directly related to therapy with the antidepressants, but it may also be related to other metabolic changes or to increased exercise as the depressive illness improves. Theophylline, a potent inhibitor of phosphodiesterase, is known to stimulate lipolysis in vitro in isolated fat cells. Theophylline also increases the intracellular concentration of cyclic AMP.* Presumably the increase in lipolysis is secondary to the increased concentrations of intracellular cyclic AMP. Amitriptyline inhibits cyclic 3’,5’AMP phosphodiesterase in heart muscle and may have similar effects on adipose tissue. As shown in Table 2, amitriptyline, like theophylline, increased the concentration of intraceullar cyclic AMP. It also markedly increased the concentration of cyclic AMP caused by DL-arterenol plus theophylline. These results are consistent with the interpretation that amitriptyline is an inhibitor of phosphodiesterase. In contrast to theophylline, however, the data in Table 1 indicate that amitriptyline does not stimulate basal lipolysis and is in fact a potent inhibitor of induced lipolysis in spite of its ability to enhance markedly the intracellular concentration of cyclic AMP. The data given in Table 2 were obtained when the isolated fat cells were incubated for 8 min. It has been demonstrated that the maximal concentration of cyclic AMP in isolated fat cells occurs within approximately 6-8 min and slowly declines with 15-20 min of incubation.* The data of Butcher et al. suggests that cyclic 3’SAMP is responsible for activating lipolysis, but the rate of lipolysis does not parallel cyclic AMP concentration beyond approximately a twofold increase over basal levels. The methyl xanthines have provided amplification of the cyclic AMP response to hormones that stimulate adenyl cyclase, thus facilitating studies not only of these hormones but also of agents that act to decrease cyclic AMP levels. It can be seen in Table 2 that after 8 min of incubation with DL-arterenol plus theophylline, there was a marked increase in the concentration of cyclic AMP and also a significant increase in the release of glycerol. Amitriptyline in the presence of DL-arterenol and theophylline gave a further increase in the concentration of cyclic AMP but no change in glycerol release. If the incubations are continued for 1 hr, however, amitriptyline, as shown in Table 1, is a potent inhibitor of lipolysis induced by DL-arterenol plus theophylline, despite the fact that it enhances the initial concentration of cyclic AMP in the fat cell. It has recently been demonstrated that both deoxyfrenolicin and Vitamin K, are antilipolytic but act synergistically with norepinephrine or corticotropin to elevate intracellular levels of cyclic AMP in isolated fat cells.22.23 It has been suggested that cyclic AMP in adipose tissue cells may be present in different compartments and all of the cyclic AMP may not be available for stimulating lipolysis. 24,25 The concentration of cyclic AMP required for the activation of lipase may be very critical and excess cyclic AMP may cause a reduction in lipase activity.26. The mechanism by which amitriptyline inhibits lipolysis and yet increases the intracellular concentration of cyclic AMP is unknown. The fact that amitriptyline can inhibit lipolysis under conditions where cyclic AMP concentration is increased suggests that the antilipolytic effect of amitriptyline
LOVRIEN
228
ETAL.
results from the inhibition of the lipolytic enzyme system that is activated by cyclic AMP, rather than the effect of amitriptyline on cyclic AMP concentration itself. The increase in the intracellular concentration of cyclic AMP caused by amitriptyline may be due to the inhibition of phosphodiesterase. It is possible that amitriptyline inhibits the binding of cyclic AMP not only to phosphodiesterase but also inhibits cyclic AMP binding to a protein kinase or triglyceride lipase. This would inhibit lipolysis by blocking the activation of triglyceride lipase and at the same time there would be an increase in intracellular concentration of cyclic AMP because of the inability of phosphodiesterase to convert the cyclic AMP to SAMP. It is also possible that amitriptyline directly inhibits triglyceride lipase. ACKNOWLEDGMENT We are grateful to Dr. Nelson Goldberg and Dr. Joseph Larner for their help in teaching us to assay cyclic AMP and helping in the preparation of phosphodiesterase. REFERENCES 1. Hollenburg, C. H.: Adipose tissue lipases. II. In Renold, A. E., and Cahill, G. F. (Eds.): Handbook of Physiology, Section 5: Adipose Tissue. Washington, D. C., American Physiology Society, 1965. 2. Rodbell, M.: The metabolism of isolated fat cells. In Renold, A. E., and Cahill, G. F. (Eds.): Handbook of Physiology, Section 5: Adipose Tissue. Washington, D. C., American Physiology Society, 1965. 3. Butcher, R. W., Ho, R. J., Meng, H. D., and Sutherland, E. W.: Adenosine 3’, 5’monophosphate in biological materials. II. The measurement of adenosine 3’, 5’-monophosphate in tissues and the role of the cyclic nucleotide in the lipolytic response of fat to epinephrine. J. Biol. Chem. 240: 4515,1965. 4.
Fain, J. N., Kovacev, V. M., and Scow, R. 0.: Antilipolytic effect of insulin in isolated fat cells of the rat. Endocrinology 78: 773, 1966. 5. Brown, J. D., and Stone, D. B.: Antilipolytic effects of sulfonylurea drugs on induced lipolysis in isolated fat cells of the rat. Endrocrinology 81:71, 1967. 6. Stone, D. B., and Brown, J. D.: In vitro effects of phenformin hydrochloride: Observations using isolated fat cells. Ann. N. Y. Acad. Sci. 148:623, 1968. 7. Brown, J. D., Stone, D. B., and Steele, A. A.: The mechanism of action of antilipolytic agents: A comparison of the effects of insulin, tolbutamide, and phenformin on lipolysis induced by dibutyryl cyclic AMP plus theophylline. Metabolism 18:926, 1969.
8. Butcher, R. W., Baird, C. E., and Suthand erland, E. W.: Effects of lipolytic antilipolytic substances on adenosine 3’, s’monophosphate levels in isolated fat cells. J. Biol. Chem. 243:1705, 1968. 9. Rodbell, M.: Metabolism of isolated fat cells. I. Effects of hormones on glucose metabolism and lipolysis. J. Biol. Chem. 239:375,1964. 10. Fain, J. N.: Effect of dexamethasone and growth hormone on fatty acid mobilization and glucose utilization in adrenalectomized rats. Endrocrinology 71:633, 1962. II. Rodbell, M.: Modulation of lipolysis in adipose tissue by fatty acid concentration in fat cell. Ann. N. Y. Acad. Sci. 131: 302, 1965.
0.: Eine Enzymatische 12. Wieland, Methode zur Bestimmung von Glycerin. Biothem.
Z. 329:313, 1957.
13. -,
and Suyter, M.: Glycerokinase: Isolierung und Eigenschaften des Enzyms. Biochem. Z. 329:330,1957. 14. Stone, D. B., Brown, J. D., and Cox, C. P.: Effect of tolbutamide and phenformin on lipolysis in adipose tissue in vitro. Amer. J. Physiol. 210:26, 1966. 15. Dole, V. I’., and Meinertz, H.: Microdetermination of long-chain fatty acid in plasma and tissues. J. Biol. Chem. 235: 2595, 1960. 16. Goldberg, N. D., Villar-Palasi, C., Sasko, H., and Larner, J.: Effects of insulin treatment on muscle 3’, 5’-cyclic adenylate levels in vivo and in vitro. Biochem. Biophys. Acta 148:665, 1967.
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17. Huntsberger, D. V., and Leaverton, P. E.: Statistical inference in the biomedical sciences. Boston, Allyn & Bacon, 1970, p. 211. 18. Corbin, J. D., and Krebs, E. G.: A cyclic AMP stimulated protein kinase in adipose tissue. Biochem. Biophys. Res. Commun. X:328, 1969. 19. Abdulla, Y. H., and Hamadah, K.: 3’, 5’-cyclic adenosine monophosphate in depression and mania. Lancet 1:378, 1970. 20. Ramsden, N.: Cyclic AMP in depression and mania. Lancet 2 :lOS,1970. 21. Paul, M. I., Kitzion, B. R., and Janowsky, D. S.: Affective illness and cyclic AMP excretion. Lancet 1:88, 1970. 22. Kuo, J. F.: Effects of deoxyfrenolicin on isolated adipose cells. II. Lipolysis adenosine 3’, 5’-monophosphate levels, and comparison with the effects of Vitamin K,. Biothem. Pharmacol. 18:757, 1969.
23. -, and Greengard, I’.: Cyclic nucleotide-dependent protein kinases. J. Biol. Chem. 243~4067, 1970. 24. Butcher, R. W., Sneyd, J. G. T., Park, C. R., and Sutherland, E. W., Jr.: Effect of insulin on adenosine 3’, 5’-monophosphate in the rat epididymal fat pad. J. Biol. Chem. 241:1651, 1966. 25. Kuo, J. F., and DeRenzo, E. C.: A comparison of the effects of lipolytic and antilipolytic agents on adenosine 3’, Smonophosphate levels in adipose cells as determined by prior labeling with adenineB-14C. J. Biol. Chem. 244 :2252,1969. 26. Rizack, M. A.: Activation of an epinephrine-sensitive lipolytic activity from adipose tissue by adenosine 3’, $-phosphate. J. Biol. Chem. 239:392, 1964.