Acta med. scand. Vol. 197, pp. 271-274, 1975
THE EFFECT OF NOREPINEPHRINE AND THEOPHYLLINE ON BLOOD GLUCOSE, PLASMA FFA, PLASMA GLYCEROL AND PLASMA INSULIN I N NORMAL SUBJECTS Krister Amman, Sven Carlstrom and Jan I . Thorell From the Department of Internal Medicine A , University Hospital, Lund. and the Isotopr Luboratory, Malmo General Hospital, Malmii, Sweden
Abstract. The plasma concentrations of free fatty acids (FFA), glycerol and insulin as well as the blood glucose concentration have been followed in two groups of subjects
after infusions of theophyllamine. Each individual was examined twice. The 5 subjects in group I were given an infusion of norepinephrine before the theophyllamine at one of the examinations and saline at the other. The 6 subjects in group I1 were given an infusion of norepinephrine at both examinations, followed by theophyllamine on one occasion and by saline on the other. Thus, the subjects in both groups served as their own controls. It was found that theophyllamine caused lipid mobilization, as measured by the plasma FFA and plasma glycerol concentrations, both when given as the only active drug and when given after norepinephrine. The blood glucose concentration rose slightly after norepinephrine and the plasma insulin level increased concomittantly. When theophylline was given as the only active drug, there was no increase in the blood glucose but the plasma insulin concentration rose slightly. Theophylline has been shown to exert a stimulating effect on the lipolytic process in adipose tissue in animals (2) and on human adipose tissue in vitro (3). According to the current theory, the lipolytic effect is mediated through inhibition of the phosphodiesterase activity, which enzyme converts cyclic A M P (CAMP)t o inactive AMP. Theophylline, thus, increases the c A M P concentration by diminishing its breakdown an d , as the cAMP concentration controls the rate of lipolysis, consequently stimulates lipolysis. The catecholamines, on the other hand, exert an effect on lipolysis by stimulation of the adrenergic receptors in adipose tissue, causing an activation of the enzyme adenylcyclase, which converts AT P t o CAMP. A s a result. the concentration of c A M P is
increased and lipolysis stimulated. Thus, theophylline and catecholamines both increase the cAMP concentration, but their effects are mediated via separate enzymatic mechanisms and it is theoretically possible t o expect an additional effect when both drugs are given. The present study was started as a trial t o elucidate: 1) if theophylline in vivo has a n effect on the lipolysis and the lipid mobilization, as measured by the plasma free fatty acid (FFA ) and the plasma glycerol concentrations, and 2) if theophylline in vivo further increases the lipid mobilization induced by norepinephrine. Preliminary results of this study have been published earlier (5). MATERIAL A N D METH O D S Eleven apparently healthy, male volunteers, aged 2 3 4 4 years, were studied. The subjects were divided into group I (n=5, mean age 27 years) and group I1 (n =6, mean age 30 years) and each individual was examined on two occasions on different days. The examination started in the morning with the subjects in the fasting state. One catheter was inserted into the brachial artery and one into one of the cubital veins. After about one hour’s rest infusions of the drugs were given via the venous catheter and blood samples were drawn through the arterial catheter. The drugs were infused during two consecutive periods of 10 min each whereafter the subjects rested for 60 min. The subjects in group I were on one occasion given 75 pg norepinephrine during the first 10 niin and 10 ml theophyllamine “ACO” (theophylline 23 mglml) during the following 10 min. On the other occasion the same individuals were given physiological saline during the first period and the theophyllamine during the second period. Thus, the individuals served as their own controls. In group I1 the subjects were given 75 pg norepinephrine Acta med. scand. 197
272
K.
ul.
Arnmun rt
during the first infusion period on both occasions. I t was followed by 10 ml theophyllamine “ACO” as the second infusion on one occasion and by physiological saline on the other. In this group, too, the subjects served as their own controls. During the whole experimental period ECG was recorded and direct measurements of the intraarterial BP were made at intervals. Blood samples were drawn at 60 and 30 min before, at the start of and at I , 3, 5 , 8 , I I , 13, 15 and 18 min during the infusions. After the infusions blood samples were drawn at 21,23,25,28,35,50,65and 80 min. The blood was collected in test tubes containing heparin as anticoagulant. The blood samples were kept in ice-water and plasma was separated within 90 min. The whole experiment was performed in the Department of Clinical Physiology, University Hospital of Lund, Sweden. Plasma FFA was titrated according to Trout et al. (13). Plasma glycerol was determined according to Laurel1 and Tibbling (9) and blood glucose according to Marks ( I I ) . Plasma insulin was estimated with the method of Heding (7).
I lni5J
Group
i p +Thw NoClr Thm
CI K
-
RESULTS
-60
-JO
0
The plasma FFA, plasma glycerol, blood glucose and plasma insulin concentrations in group I during the experiment are illustrated in Fig. 1. I t is apparent, that the norepinephrine and theophylline infusions increased the concentrations of plasma FFA, plasma glycerol and blood glucose. The plasma insulinlevel rose slightly, parallel tothe increase in blood glucose. When the individuals in group I were given saline as the first infusion, the following theophylline infusion caused a rise in the plasma FFA and the plasma glycerol, blood glucose and plasma insulin concentraitions in group 11. When norepinephrine was given showed no variations. The plasma insulin level rose slightly but significantly. Fig. 2 shows the mean plasma FFA, plasma
5
Hour3
Fig. I . Plasma FFA. plasma glycerol, blood glucose and plasma insulin concentrations during the experiment in group 1. Mean fS.E.M.
Table I . Hemodynarnic data ~~~
Combinations of infusions
~~~
~~
Before infusion
During infusion I
During infusion I1
After infusion
BP
BP
BP
BP
S
D
M
H
R
S
D
M
H
R
S
D
M
H
R
S
D
M
H
R
Group 1 (n =5)
N-ep+Theo NaCI+ The0
116 71 114 67
91 85
60 52
143 83 116 66
85
108 48 53
123 68 122 71
90 90
64 56
122 73 126 73
93 96
62 61
109 68 1 1 1 65
82 83
56 53
135 69 131 78
103 49 100 46
114 66 115 62
84 83
60 55
115 64 114 67
87 83
58 56
Group 11 ( n =6)
N-ep+ The0 N-ep+NaCl Acra med. scand. 197
Norepinephrine and rheophylline studies
273
The hemodynamic data are summarized in Table I. In group I the mean intraarterial BP rose and the heart rate decreased when norepinephrine was given as the first infusion. When saline was given, no variations were noticed. The subjects in group I1 showed on both occasions a rise in the mean arterial BP and a bradycardia following the norepinephrine infusion. The theophylline infusion caused no variations in the hemodynamic parameters in either group. DISCUSSION
W R S
nm,
1”
m,wm
Fig. 2. Plasma FFA, plasma glycerol, blood glucose and plasma insulin concentrations during the experiment in group 11. Mean +S.E.M.
glycerol, blood glucose and plasma insulin concentrations in group IT. When norepinephrine was given as the only active drug, followed by saline, there were rises in plasma FFA, plasmaglycerol and blood glucose of the same magnitude as in group I with the same infusions. When, however, the norepinephrine infusion was followed by theophylline, the rises in plasma FFA and plasma glycerol were somewhat more pronounced, lasted longer and showed a tendency towards two maxima. The blood glucose curve was the same on both occasions in group 11. Both curves showed a slight rise and a concomitant slight increase in the plasma insulin level. 18-752984
These studies indicate that theophyllamine is an active stimulator oflipolysis and lipid mobilization in vivo when measured as the plasma concentrations of FFA and glycerol. The response to theophyllamine was delayed as compared to the norepinephrine response. This supports the view of dissimilar mechanisms for the effect of norepinephrine and theophyllamine on the lipolysis. If one accepts the theory that their effects are related to an increased production rate of adenylcyclase by the former and a decreased degradation rate by the latter, one would expect the combined use of both agents to result in exaggerated responses regarding FFA and glycerol. This was the case and furthermore, these combined responses had a 2-phase character, as if the two stimulants worked in an unrelated manner. Theophyllamine given as the only drug induced a small rise in insulin in spite of the absence of changes in blood glucose. It is known that agents which raise the concentration of cAMP in the B cell enhance the glucose-stimulated release of insulin (lo), though this effect is not seen in the absence of glucose. But the marked increase in FFA may have exerted an influence as well. It has been reported that experimental elevation of FFA in dogs stimulates the release of insulin (6). In man FFA alone does not stimulate to increased insulin secretion (12) but may enhance the response to other stimuli (1). Combined enhancing effects of cAMP and FFA may therefore cause a sensitivation of the B cells for glucose to the extent that the basal concentration of glucose is high enough to induce an increased release of insulin. The even higher insulin response noted in the norepinephrine experiments when the glucose level rose slightly, is then probablyjust an exaggeration of the same effect. The lipolytic response, as well as the hemodynamic data, when norepinephrine was given Acta med. scand. 197
274
K . Arnman et al.
as the only active drug, were in agreement with earlier findings (4). Theophylline showed no hemodynamic effects. Theophylline is commonly used in the treatment of asthma bronchiale and cardiac insufficiency. Judging from the present results it is able to cause an elevation of the plasma FFA concentration. Kurien et al. (8) have proposed that raised plasma FFA levels may induce arrhythmias in patients with myocardial infarction. If their theory is correct, theophylline should perhaps be avoided in the treatment of cardiac insufficiency in patients with myocardial injury. ACKNOWLEDGEMENTS Financial support was obtained from the Swedish Medical Research Council (grants no. K73-19X-3784 and 13X3508), the Medical Faculty, University of Lund, and the National Swedish Association against Heart and Chest Diseases.
REFERENCES Balasse, E. 0. & Ooms, H. A.: Role of plasma free fatty acids in the control of insulin secretion in man. Diabetologia 9: 145, 1973. Brodie, B. B., Davies, J. I., Hynie, S., Krishna, G. & Weiss, B.: Interrelationships of catecholamines with other endocrine systems. Pharm. Rev. 18: 273, 1966. Carlsson, L. A., Hallberg, D. & Micheli, H.: Quantitative studies on the lipolysis response of human adipose tissue to noradrenaline and theophylline. Acta med. scand. 185:465, 1969.
Acta med. scand. 197
4. Carlstrom, S.: Studies on fatty acid metabolism in diabetics during exercise. V1. Infusions of norepinephrine to male, non-insulin treated, juvenile diabetics. Acta med. scand. 182 513, 1967. 5. Carlstrom, S. & Thorell, J.: The effect of norepinephrine and theophylline on blood glucose, plasma FFA, plasma glycerol and plasma insulin in normal subjects. Excerpta med., International Congress Series No. 280 95, 1973. 6. Crespin, S. R., Greenough 111, W. B. & Steinberg, D.: Stimulation of insulin secretion by infusion of free fatty acids. J. clin. Invest. 48: 1934, 1%9. 7. Heding, L. G.: A simplified insulin radioimmunoassay method. In: Labeled protein in tracer studies (ed. L. Danato, G . Milhaud & F. Suchis), pp. 345-351. Euratom 1966. 8. Kurien, V. A., Yates, P. A. & Olivier, M.F.: Free fatty acids, heparin and arrhythmias during experimental myocardial infarction. Lancet 2: 185, 1969. 9. Laurell, S. & Tibbling, G.: An enzymatic fluorometric micromethod for the determination of glycerol. Clin. chim. Acta 13: 317, 1966. 10. Malaisse, W. J . , Malaisse-Lagae, F. & Mayhew, D.: A possible role for the adenylcyclase system in the insulin secretion. J. clin. Invest. 46: 1724, 1%7. 1 I . Marks, V.: An improved glucose-oxidase method for determining blood, CSF and urine glucose levels. Clin. chim. Acta 4: 395, 1959. 12. Schalch, D. S. & Kipnis, D. M.: Abnormalities in carbohydrate tolerance associated with elevated plasma nonesterified fatty acids. J. clin. Invest. 44: 2010, 1965. 13. Trout, D. L . , Estes, E. H., Jr & Friedberg. S. J.: Titration of free fatty acids of plasma: A study of current methods and a new modification. J. Lipid Res. 1: 199, 1960.
THE EFFECT OF NOREPINEPHRINE AND THEOPHYLLINE ON BLOOD GLUCOSE, PLASMA FFA, PLASMA GLYCEROL AND PLASMA INSULIN I N NORMAL SUBJECTS Krister Amman, Sven Carlstrom and Jan I . Thorell From the Department of Internal Medicine A , University Hospital, Lund. and the Isotopr Luboratory, Malmo General Hospital, Malmii, Sweden
Abstract. The plasma concentrations of free fatty acids (FFA), glycerol and insulin as well as the blood glucose concentration have been followed in two groups of subjects
after infusions of theophyllamine. Each individual was examined twice. The 5 subjects in group I were given an infusion of norepinephrine before the theophyllamine at one of the examinations and saline at the other. The 6 subjects in group I1 were given an infusion of norepinephrine at both examinations, followed by theophyllamine on one occasion and by saline on the other. Thus, the subjects in both groups served as their own controls. It was found that theophyllamine caused lipid mobilization, as measured by the plasma FFA and plasma glycerol concentrations, both when given as the only active drug and when given after norepinephrine. The blood glucose concentration rose slightly after norepinephrine and the plasma insulin level increased concomittantly. When theophylline was given as the only active drug, there was no increase in the blood glucose but the plasma insulin concentration rose slightly. Theophylline has been shown to exert a stimulating effect on the lipolytic process in adipose tissue in animals (2) and on human adipose tissue in vitro (3). According to the current theory, the lipolytic effect is mediated through inhibition of the phosphodiesterase activity, which enzyme converts cyclic A M P (CAMP)t o inactive AMP. Theophylline, thus, increases the c A M P concentration by diminishing its breakdown an d , as the cAMP concentration controls the rate of lipolysis, consequently stimulates lipolysis. The catecholamines, on the other hand, exert an effect on lipolysis by stimulation of the adrenergic receptors in adipose tissue, causing an activation of the enzyme adenylcyclase, which converts AT P t o CAMP. A s a result. the concentration of c A M P is
increased and lipolysis stimulated. Thus, theophylline and catecholamines both increase the cAMP concentration, but their effects are mediated via separate enzymatic mechanisms and it is theoretically possible t o expect an additional effect when both drugs are given. The present study was started as a trial t o elucidate: 1) if theophylline in vivo has a n effect on the lipolysis and the lipid mobilization, as measured by the plasma free fatty acid (FFA ) and the plasma glycerol concentrations, and 2) if theophylline in vivo further increases the lipid mobilization induced by norepinephrine. Preliminary results of this study have been published earlier (5). MATERIAL A N D METH O D S Eleven apparently healthy, male volunteers, aged 2 3 4 4 years, were studied. The subjects were divided into group I (n=5, mean age 27 years) and group I1 (n =6, mean age 30 years) and each individual was examined on two occasions on different days. The examination started in the morning with the subjects in the fasting state. One catheter was inserted into the brachial artery and one into one of the cubital veins. After about one hour’s rest infusions of the drugs were given via the venous catheter and blood samples were drawn through the arterial catheter. The drugs were infused during two consecutive periods of 10 min each whereafter the subjects rested for 60 min. The subjects in group I were on one occasion given 75 pg norepinephrine during the first 10 niin and 10 ml theophyllamine “ACO” (theophylline 23 mglml) during the following 10 min. On the other occasion the same individuals were given physiological saline during the first period and the theophyllamine during the second period. Thus, the individuals served as their own controls. In group I1 the subjects were given 75 pg norepinephrine Acta med. scand. 197
272
K.
ul.
Arnmun rt
during the first infusion period on both occasions. I t was followed by 10 ml theophyllamine “ACO” as the second infusion on one occasion and by physiological saline on the other. In this group, too, the subjects served as their own controls. During the whole experimental period ECG was recorded and direct measurements of the intraarterial BP were made at intervals. Blood samples were drawn at 60 and 30 min before, at the start of and at I , 3, 5 , 8 , I I , 13, 15 and 18 min during the infusions. After the infusions blood samples were drawn at 21,23,25,28,35,50,65and 80 min. The blood was collected in test tubes containing heparin as anticoagulant. The blood samples were kept in ice-water and plasma was separated within 90 min. The whole experiment was performed in the Department of Clinical Physiology, University Hospital of Lund, Sweden. Plasma FFA was titrated according to Trout et al. (13). Plasma glycerol was determined according to Laurel1 and Tibbling (9) and blood glucose according to Marks ( I I ) . Plasma insulin was estimated with the method of Heding (7).
I lni5J
Group
i p +Thw NoClr Thm
CI K
-
RESULTS
-60
-JO
0
The plasma FFA, plasma glycerol, blood glucose and plasma insulin concentrations in group I during the experiment are illustrated in Fig. 1. I t is apparent, that the norepinephrine and theophylline infusions increased the concentrations of plasma FFA, plasma glycerol and blood glucose. The plasma insulinlevel rose slightly, parallel tothe increase in blood glucose. When the individuals in group I were given saline as the first infusion, the following theophylline infusion caused a rise in the plasma FFA and the plasma glycerol, blood glucose and plasma insulin concentraitions in group 11. When norepinephrine was given showed no variations. The plasma insulin level rose slightly but significantly. Fig. 2 shows the mean plasma FFA, plasma
5
Hour3
Fig. I . Plasma FFA. plasma glycerol, blood glucose and plasma insulin concentrations during the experiment in group 1. Mean fS.E.M.
Table I . Hemodynarnic data ~~~
Combinations of infusions
~~~
~~
Before infusion
During infusion I
During infusion I1
After infusion
BP
BP
BP
BP
S
D
M
H
R
S
D
M
H
R
S
D
M
H
R
S
D
M
H
R
Group 1 (n =5)
N-ep+Theo NaCI+ The0
116 71 114 67
91 85
60 52
143 83 116 66
85
108 48 53
123 68 122 71
90 90
64 56
122 73 126 73
93 96
62 61
109 68 1 1 1 65
82 83
56 53
135 69 131 78
103 49 100 46
114 66 115 62
84 83
60 55
115 64 114 67
87 83
58 56
Group 11 ( n =6)
N-ep+ The0 N-ep+NaCl Acra med. scand. 197
Norepinephrine and rheophylline studies
273
The hemodynamic data are summarized in Table I. In group I the mean intraarterial BP rose and the heart rate decreased when norepinephrine was given as the first infusion. When saline was given, no variations were noticed. The subjects in group I1 showed on both occasions a rise in the mean arterial BP and a bradycardia following the norepinephrine infusion. The theophylline infusion caused no variations in the hemodynamic parameters in either group. DISCUSSION
W R S
nm,
1”
m,wm
Fig. 2. Plasma FFA, plasma glycerol, blood glucose and plasma insulin concentrations during the experiment in group 11. Mean +S.E.M.
glycerol, blood glucose and plasma insulin concentrations in group IT. When norepinephrine was given as the only active drug, followed by saline, there were rises in plasma FFA, plasmaglycerol and blood glucose of the same magnitude as in group I with the same infusions. When, however, the norepinephrine infusion was followed by theophylline, the rises in plasma FFA and plasma glycerol were somewhat more pronounced, lasted longer and showed a tendency towards two maxima. The blood glucose curve was the same on both occasions in group 11. Both curves showed a slight rise and a concomitant slight increase in the plasma insulin level. 18-752984
These studies indicate that theophyllamine is an active stimulator oflipolysis and lipid mobilization in vivo when measured as the plasma concentrations of FFA and glycerol. The response to theophyllamine was delayed as compared to the norepinephrine response. This supports the view of dissimilar mechanisms for the effect of norepinephrine and theophyllamine on the lipolysis. If one accepts the theory that their effects are related to an increased production rate of adenylcyclase by the former and a decreased degradation rate by the latter, one would expect the combined use of both agents to result in exaggerated responses regarding FFA and glycerol. This was the case and furthermore, these combined responses had a 2-phase character, as if the two stimulants worked in an unrelated manner. Theophyllamine given as the only drug induced a small rise in insulin in spite of the absence of changes in blood glucose. It is known that agents which raise the concentration of cAMP in the B cell enhance the glucose-stimulated release of insulin (lo), though this effect is not seen in the absence of glucose. But the marked increase in FFA may have exerted an influence as well. It has been reported that experimental elevation of FFA in dogs stimulates the release of insulin (6). In man FFA alone does not stimulate to increased insulin secretion (12) but may enhance the response to other stimuli (1). Combined enhancing effects of cAMP and FFA may therefore cause a sensitivation of the B cells for glucose to the extent that the basal concentration of glucose is high enough to induce an increased release of insulin. The even higher insulin response noted in the norepinephrine experiments when the glucose level rose slightly, is then probablyjust an exaggeration of the same effect. The lipolytic response, as well as the hemodynamic data, when norepinephrine was given Acta med. scand. 197
274
K . Arnman et al.
as the only active drug, were in agreement with earlier findings (4). Theophylline showed no hemodynamic effects. Theophylline is commonly used in the treatment of asthma bronchiale and cardiac insufficiency. Judging from the present results it is able to cause an elevation of the plasma FFA concentration. Kurien et al. (8) have proposed that raised plasma FFA levels may induce arrhythmias in patients with myocardial infarction. If their theory is correct, theophylline should perhaps be avoided in the treatment of cardiac insufficiency in patients with myocardial injury. ACKNOWLEDGEMENTS Financial support was obtained from the Swedish Medical Research Council (grants no. K73-19X-3784 and 13X3508), the Medical Faculty, University of Lund, and the National Swedish Association against Heart and Chest Diseases.
REFERENCES Balasse, E. 0. & Ooms, H. A.: Role of plasma free fatty acids in the control of insulin secretion in man. Diabetologia 9: 145, 1973. Brodie, B. B., Davies, J. I., Hynie, S., Krishna, G. & Weiss, B.: Interrelationships of catecholamines with other endocrine systems. Pharm. Rev. 18: 273, 1966. Carlsson, L. A., Hallberg, D. & Micheli, H.: Quantitative studies on the lipolysis response of human adipose tissue to noradrenaline and theophylline. Acta med. scand. 185:465, 1969.
Acta med. scand. 197
4. Carlstrom, S.: Studies on fatty acid metabolism in diabetics during exercise. V1. Infusions of norepinephrine to male, non-insulin treated, juvenile diabetics. Acta med. scand. 182 513, 1967. 5. Carlstrom, S. & Thorell, J.: The effect of norepinephrine and theophylline on blood glucose, plasma FFA, plasma glycerol and plasma insulin in normal subjects. Excerpta med., International Congress Series No. 280 95, 1973. 6. Crespin, S. R., Greenough 111, W. B. & Steinberg, D.: Stimulation of insulin secretion by infusion of free fatty acids. J. clin. Invest. 48: 1934, 1%9. 7. Heding, L. G.: A simplified insulin radioimmunoassay method. In: Labeled protein in tracer studies (ed. L. Danato, G . Milhaud & F. Suchis), pp. 345-351. Euratom 1966. 8. Kurien, V. A., Yates, P. A. & Olivier, M.F.: Free fatty acids, heparin and arrhythmias during experimental myocardial infarction. Lancet 2: 185, 1969. 9. Laurell, S. & Tibbling, G.: An enzymatic fluorometric micromethod for the determination of glycerol. Clin. chim. Acta 13: 317, 1966. 10. Malaisse, W. J . , Malaisse-Lagae, F. & Mayhew, D.: A possible role for the adenylcyclase system in the insulin secretion. J. clin. Invest. 46: 1724, 1%7. 1 I . Marks, V.: An improved glucose-oxidase method for determining blood, CSF and urine glucose levels. Clin. chim. Acta 4: 395, 1959. 12. Schalch, D. S. & Kipnis, D. M.: Abnormalities in carbohydrate tolerance associated with elevated plasma nonesterified fatty acids. J. clin. Invest. 44: 2010, 1965. 13. Trout, D. L . , Estes, E. H., Jr & Friedberg. S. J.: Titration of free fatty acids of plasma: A study of current methods and a new modification. J. Lipid Res. 1: 199, 1960.
