Pflfigers Arch. 369, 21
Pflfigers Archiv
25 (1977)
European Journal of Physiology 9 by Springer-Verlag 1977
Lack of Influence of Glucagon on Glucose Homeostasis after Prolonged Exercise in Rats H. GALBO, E. A. RICHTER, J. J. HOLST, and N. J. CHRISTENSEN Institute of Medical Physiology B, University of Copenhagen, DK-2100 Copenhagen, Department of Clinical Chemistry, Bispebjerg Hospital, DK-2400 Copenhagen Second Clinic of Internal Medicine, Kommunehospitalet, DK-8000, •rhus, Denmark
Summary. The significance of glucagon for postexercise glucose homeostasis has been studied in rats fasted overnight. Immediately after exhaustive swimming either rabbit-antiglucagon serum or normal rabbit serum was injected by cardiac puncture. Cardiac blood and samples of liver and muscle tissue were collected before exercise and repeatedly during a 120 min recovery period after exercise. During the post-exercise period plasma glucagon concentrations decreased but remained above pre-exercise values in rats treated with normal serum, while rats treated with antiglucagon serum had excess antibody in plasma throughout. Nevertheless, all other parameters measured showed similar changes in the two groups. Thus after exercise the grossly diminished hepatic glycogen concentrations remained constant, while the decreased blood glucose concentrations were partially restored. Simultaneously concentrations in blood and serum of the main gluconeogenic substrates, lactate, pyruvate, alanine and glycerol declined markedly. During the post-exercise period NEFA concentrations in serum and plasma insulin concentrations remained increased and decreased, respectively, while plasma catecholamines did not differ from basal values. Muscle glycogen concentrations decreased slightly. These findings suggest that in the recovery period after exhaustive exercise the increased glucagon concentrations in plasma do not influence gluconeogenesis.
known to imply activation of gluconeogenesis [5, 7, 17, 19]. Since it is clear from many experiments with preparations of rat liver that glucagon can stimulate gluconeogenesis strongly [ 5 - 7 , 17] this leads to the general supposition that such an effect occurs in vivo. However, definitive experiments with regard to the role of glucagon in control of gluconeogenesis in vivo have not yet been carried out. In the present study antiglucagon antibodies were administered to rats immediately after exhaustive swimming in order to evaluate the influence of endogenous pancreatic glucagon on glucose homeostasis in the post-exercise period.
MATERIALS AND METHODS
INTRODUCTION
51 male, 10-- 12 h fasted Wistar rats were used in the experiments. Eight rats were resting controls, while 43 rats weighing 2 5 0 - 270 g were forced to swim with a tail weight (4 ~ of body weight) in water maintained at 3 3 - 34~C until exhaustion. At termination of exercise, the animals were immediately anesthetized with ether. 10 rats were then sacrificed, while in 33 rats 0.2 ml of blood were drawn by cardiac puncture for glucose analysis. Then 0.5 ml of either normal rabbit serum (N-rats, n -- 18) or rabbit-antiglucagonserum (A-rats, n = 15) were injected through the cardiac cannula and the rats were placed in cages with access to water, but not to food. 30 rain after exercise 9 N-rats and 7 A-rats were sacrificed. 120 rain after exercise 9 N-rats and 8 A-rats were sacrificed. 30 and 60 min post-exercise these rats were superficially anesthetized with ether and had 0.2 ml of blood drawn by cardiac puncture for glucose analysis. At sacrifice all animals were anesthetized with ether and 6 - 8 ml of blood (A- and N-rats, respectively) were drawn into heparinized syringes for analysis of hormones and substrates 1. Samples of the liver, of the superficial part of the vastus lateralis muscle (which consists predominantly of fast-twitch white fibers [2]), of the deep portion of the vastus lateralis muscle (predominantly fast-twitch red fibers [2]), and of the soleus muscle (predominantly slow-twitch red fibers [2]) were quickly removed and frozen in liquid nitrogen.
Glucagon secretion is enhanced during brief fasting [7, 17], certain forms of diabetes [7, 17], and during the recovery period after exercise [10], conditions
1 Serial cardiac puncture and ether anesthesia has previously been shown to increase plasma glucose concentrations, whereas insulin was not changed [3]
Key words." Insulin -
Epinephrine phrine - Glycogen - Gluconeogenesis.
Norepine-
22
Pflfigers Arch. 369 (1977)
Table 1. The influence on liver and muscle glycogen (mmoles of glucose/kg of wet tissue) of exercise and of administration immediately after exercise of antiglucagon-antiserum Before exercise
0 min postexercise
30 rain postexercise
120 min postexercise
Liver
354 _+ 26 (8)
12 i 2 + (10)
A-rats N-rats
13 • i + 16 • 3 +
(7) (9)
11 • i + 12 4- 1 +
Superficialvastus
33 _+ 2
(8)
17 + 2 + (10)
A-rats N-rats
14 • 2 + 14 • 3 +
(6) (8)
11 • 2 § (8) 8 4- 2 +* (8)
Deepvastus
23
_+ 2
(8)
12 4- 2 + (10)
A-rats N-rats
11 + 1 + 12 + 2 +
(7) (9)
10 • 3 + 8 • 1+
(7) (9)
Soleus
18
4- 1
(8)
18 4- 2
A-rats N-rats
14 • 1 + (7) 12 • I +* (9)
13 • 2 + 14 • I +
(8) (9)
(9)
(7) (9)
Values are mean +_ S.E. N u m b e r of observations are shown in parenthesis. A- and N-rats were treated with rabbit antiglucagon-antiserum or normal rabbit serum, respectively. + and * denote that values are significantly different from values before exercise and values immediately postexercise (0 min postexercise), respectively
Blood pyruvate was determined by an enzymatic fluorimetric method [16], and plasma alanine was determined by the alan• dehydrogenase method [20]. Epinephrine and norepinephrine were determined by a previously published radio-enzymatic assay [4] modified for m e a s u r e m e n t of very small amounts. Insulin was determined by radioimmunoassay [1]. The detection limit for the assay system was 0.15 pmol/1. The methods of sampling and analysis of blood and tissue for glucagon, antiglucagon-antibody, glucose, lactate, N E F A (non-ester• fatty acids), glycerol, and glycogen have been reported elsewhere [9]. All analyses of a h o r m o n e were carried out in a single assay run. The characteristics of the antiglucagon-serum, which was administered to the rats, has been described in detail elsewhere [13]. The statistical evaluation of the data was made by t-test using the test for paired data when applicable. Differences of m e a n s were considered statistically significant when P < 0.05.
RESULTS
GLUCOSE mmol I [ 6 5
[ SWIMMING
I
Injection of i, Antig(ucagon t antiserum
k
I Norm~ rabbit serum
/, 3 2
I
I
-90
0
GLUCAGON prnol / I 700
I
I
30
60
I 120
i I i
T
500
The Exercise Period. The duration of swimming was
92 • 3 (S.E.) rain (n = 43), and did not differ significantly between rats sacrificed immediately, 30 or 120 min after exercise. During exercise a marked diminution of the hepatic glycogen content (Table 1) was accompanied by a decline in blood glucose concentrations (Fig. 1) which was identical in the various groups of swimming rats. Simultaneously, the glycogen concentrations in the deep and superficial part of the vastus muscle were halved (Table 1). The concentrations of NEFA and glycerol in serum and of lactic acid in blood increased, while the concentrations of pyruvate and alanine in plasma did not change significantly (Fig. 2). During exercise the plasma concentrations of glucagon increased 8-fold, while plasma concentrations of insulin decreased dramatically (Fig. 1). Immediately after exercise, glucose concentrations correlated significantly with glucagon concentrations (r = - 0.96, P < 0.001), but not with insulin concentrations.
300 100 I
I
-90
0
I
1
30
120
INSULINI
pmol / I 60
20
i \
5
I - 90
,i . . . .
~ 1 =::.1 0
30
rnin
. . . . =----!I 120
postexercise
Fig. 1. The concentrations of glucose in blood and of glucagon and insulin in plasma during and after exhaustive exercise. (7 pmol 9 1-1 of insulin = 1 g U - m l - 9
H. Galbo et al. : Glucagon and Post Exercise Glucose Homeostasis LACTATE mmol / I
GLYCEROL mmol / l
[ SWIMMI[~ I
0.4
? Antiglucagon ] ant iserurn ~ or Normal rabbit"J serum* I
-90
"~ 0.2 J
30
-90
I
1
0
30
I
120
NEFA meq / !. 1,6
t'-,,
1.2
too ,,
I
i I
-90
l
120
i
5o
J i
+
J
0
.I
0.3
PYRUVATE jJmol/[ 15o
23
0
[
9
30
0.8 0.4
120
t !
-90
600 t ALAN]NE [ pmo[ / l
I
I
|
0
30
120
min
postexercise
400 ~,
-.'.
-.-,.l
1
200
J
i I
-90
&
1
30
The Post-Exercise Period. After exercise, except for plasma glucagon concentrations, the course of the different variables was identical whether antiglucagonserum or normal serum had been administered (Figs. 1 and 2, Tables 1 and 2). Throughout the post-exercise period the hepatic glycogen concentrations remained at the low levels reached at exhaustion while muscle glycogen concentrations tended to decrease further (Table 1). Blood glucose concentrations initially increased but from 30 to 120 rain post-exercise blood glucose concentrations remained constant 20 % below pre-exercise values. While in the postexercise period blood glucose concentrations thus were partially restored the concentrations of the gluconeogenic substrates lactate, pyruvate, alanine, and glycerol gradually declined (Fig. 2). 30, 120, and 120 rain after exercise, respectively, the concentrations of pyruvate and lactate in blood and of alanine in plasma were significantly below pre-exercise levels. 120 rain after exercise the concentrations of glycerol in serum no longer significantly exceeded pre-exercise
Fig. 2. The concentrations of lactate and pyruvate in blood, of alanine in plasma, and of glycerol and N E F A in serum during and after exhaustive exercise
Table 2. The plasma concentrations of epinephrine and norepinephrine in rats before exercise and 30 rain after exercise. Exercised rats were treated with either rabbit antiglucagon-antiserum (A-rats) or normal rabbit serum (N-rats) immediately after exercise. Values are mean 4- S.E. Number of observations are shown in parenthesis Before exercise
30 rnin postexercise A-rats
N-rats
Epinephrine (ng/ml)
0.46 4- 0.10 (4)
0.93 _+ 0.35 (5)
0.96 4- 0.33 (4)
Norepinephrine (ng/ml)
0.93 4- 0.33 (4)
1.12 _+ 0.30 (5)
0.72 4- 0.15 (5)
values, whereas NEFA concentrations still were markedly increased (Fig. 2). Thirty minutes after exercise the plasma concentrations of epinephrine and norepinephrine did not differ significantly from concentrations at rest
24 (Table 2). Insulin concentrations in plasma, however, initially increased slightly (P < 0.01), but stayed far below pre-exercise values throughout the post-exercise period (Fig. 1). In rats treated with normal serum the glucagon concentrations in plasma decreased markedly during the first 30 min of the post-exercise period but then glucagon concentrations remained constant at about twice the pre-exercise values (Fig. 1). All rats treated with antiglucagon-serum had "excess" antibody [9, 13] in plasma at sacrifice. The "titer" [9, 13] varied from 1:500 to 1:2000.
DISCUSSION In the present study of the recovery phase after exercise the grossly diminished hepatic glycogen content was constant. Accordingly after exercise the glucose necessary not only for the metabolism of the obligatory glycolytic tissues but also for the partial restoration of extracellular glucose concentrations was produced entirely by gluconeogenesis. At rest in the post absorptive state, however, only a minor part (25 ~) of hepatic glucose production is derived from gluconeogenesis [19]. In addition, the major non obligatory glycolytic tissues (muscle [19] and adipose tissue) only consume small amounts of glucose in the basal state. Thus, even though decreased glucose uptake in non obligatory glycolytic tissues in the post-exercise period-however in contrast to the previous finding of increased muscular glucose uptake after exercise [19]-can not be excluded due to lack of direct flux determinations, available evidence strongly suggests that in the present study gluconeogenesis after exercise was increased compared with the preexercise period. While gluconeogenesis probably increased after exercise, the concentrations of lactate and pyruvate in blood and of alanine in plasma declined below preexercise levels supporting previous findings [19] of increased fractional extraction of these main gluconeogenic substrates [7] in the post-exercise period. Since glucagon concentrations in plasma remain elevated above basal levels in the immediate postexercise period (Fig. 1) [10] increased glucagon secretion has been suggested to be of importance for the accellerated rate of hepatic gluconeogenesis in the recovery phase after exercise [19]. We have recently shown that administration of antiglucagonserum to rats ensures a rapid and extensive neutralization of endogenous glucagon during a period of time comparable to the post-exercise period in the present study [13]. Nevertheless, in the present study, after exercise tissue glycogen and blood glucose concentrations were identical whether antiglucagon-serum
Pfliigers Arch. 369 (1977) or normal serum had been administered. The lack of difference in gluconeogenesis between rats treated with antiglucagon-serum and rats treated with normal serum can neither be explained by differences in the hepatic supply of essential substrates (Fig. 2) [7], nor by differences in the plasma concentrations of insulin (Fig. 1) and catecholamines (Table 2), hormones which might rapidly influence the rate of gluconeogenesis [5-7, 17]. It appears from these results that the increased concentrations of glucagon in plasma do not significantly influence hepatic glucose production in the post-exercise period, a finding which contrasts with the previously reported observation that during exercise neutralization of glucagon diminishes hepatic glucose production [9]. The discrepancy may be explained by larger glucagon concentrations during than after exercise (Fig. 1), by larger sensitivity of glycogenolysis than of gluconeogenesis to stimulation by glucagon [17] and by larger binding of glucagon and larger glucagon-stimulated cyclic AMP accumulation and glucagon-stimulated glucose production in fed than in glycogen deprived hepatic cells [5,6,8]. Furthermore, since the hyperglycemic effect of glucagon is highly dependent upon insulinopenia [12, 17, 18] the somewhat larger insulin concentrations after than during exercise (Fig. 1) possibly minimized the influence of glucagon on carbohydrate metabolism. At present we have to propose that induction during exercise ofgluconeogenic enzymes [7] possibly brought about by increased release of lactate [7], glucocorticolds [7, 14, 15], and glucagon [17] partly accounts for the increased rate of gluconeogenesis in the recovery phase after exercise. Sometimes increased hepatic supply of substrates in the post-exercise period [19] may contribute to the increased glucose formation [7], and sometimes low insulin concentrations may do so [6, 7]. After non-exhaustive exercise increased concentrations of insulin as well as of glucose have been found [19], but after exhaustive exercise in the present study the concentrations of these substances were below basal levels (Fig. 1). When glucose concentrations 30 min after exercise approached basal levels, glucagon concentrations simultaneously declined markedly while insulin concentrations remained almost constant (Fig. 1), possibly reflecting the fact that at low glucose levels the pancreatic alpha cell is more sensitive than the beta cell to changes in glucose concentrations [11]. The finding of similar rates of gluconeogenesis and of similar plasma substrate concentrations (Fig. 2) in rats treated with antiglucagon-serum compared to rats treated with normal serum indicates that glucagon does not influence peripheral metabolism in the postexercise period. Thus it appears that glucagon is not indispensable for the mobilization of free fatty acids
H. Galbo et al. : Glucagon and Post-Exercise Glucose Homeostasis
25
neither after exercise nor, as shown previously [9,15] during exercise. Low insulin concentrations (Fig. 1), on the other hand, may have decreased the uptake and oxidation of glucose in the muscles and may accordingly have contributed to the low concentrations in blood and serum of pyruvate, lactate and alanine [19]. Furthermore, the low insulin concentrations probably promoted lipolysis and also thereby served to preserve blood glucose concentrations in spite of a probably increased total metabolic rate [10] in the recovery period after exercise. The present study has shown that in rats fasting during recovery after prolonged exhausting exercise, extracellular glucose concentrations are partially restored even though hepatic glycogen concentrations are "not further diminished. The increased glucagon concentrations in plasma found during the postexercise period do not significantly influence glucose production in this period.
6. Davidson, M. J., Bertiner, J. A. : Acute effects of insulin on carbohydrate metabolism in rat liver slices: independence from glucagon. Amer. J. Physiol. 227, 7 9 - 8 7 (1974) 7. Exton, J. H. : Gluconeogenesis. Metabolism 21, 945 - 990 (1972) 8. Fouchereau-Peron, M., Rancon, F., Freychet, P., Rosselin, G. : Effect of feeding and fasting on the early steps of glucagon action in isolated rat liver cells. Endocrinology 98, 755-760 (1976) 9. Galbo, H., Holst, J. J.: The influence of glucagon on hepatic glycogen mobilization in exercising rats. Pflfigers Arch. 363, 4 9 - 53 (19761 10. Galbo, H , Holst, J.J., Christensen, N.J., Hilsted, J.: Glucagon and plasma catecholamines during beta-receptor blockade in exercising man. J. appl. Physiol. 40, 855-863 (1976) 11. Gerich, J. E., Charles, M. A., Grodsky, G. M.: Characterization of the effects of arginine and glucose on glucagon and insulin release from the perfused rat pancreas. J. clin. Invest. 54, 833-841 (1974) 12. Gerich, J. E., Lorenzi, M,, Bier, D. M., Tsalikian, E., Schneider, V., Karam, J. H., Forsham, P. H. : Effects of physiologic levels of glucagon and growth hormone on human carbohydrate and lipid metabolism. J. clin. Invest. 57, 875-884 (1974) 13. Holst, J. J., Galbo, H., Richter, E. A.: Neutralization of glucagon by antibodies. (To be submitted) 14. Huibregtse, C. A., Brunsvold, R. A., Ray, P. D.: Dietary and hormonal regulation of some enzyme activities associated with gluconeogenesis in rabbit liver. Biochim. biophys. Acta (Amst.) 421, 228 - 236 (1976) 15. Luyckx, A. S., Dresse, A., Cession-Fossion, A., Lefebvre, P. J. : Catecholamines and exercise-induced glucagon and fatty acid mobilization in the rat. Amer. J. Physiol. 229, 376-383 (1975) 16. Olsen, C.: An enzymatic fluorimetric micromethod for the determination of acetoacetate, fl-hydroxybutyrate, pyruvate and lactate. Clin. chim. Acta 33, 293-300 (1971) 17. Park, C. R., Exton, J. H.: Glucagon and the metabolism of glucose. In: Glucagon (P. J. Lefebvre and R. H. Unger, eds.) Oxford: Pergamon Press 1972 18. Sherwin, R. S., Fisher, M., Hendler, R., Felig, P.: Hyperglucagonemia and blood glucose regulation in normal, obese and diabetic subjects. New Engl. J. Med. 294, 455-461 (1976) 19. Wahren, J., Felig, P., Hendler, R., Ahlborg, G.: Glucose and amino acid metabolism during recovery after exercise. J. appl. Physiol. 34, 838-845 (1973) 20. Williamson, D. H.: Methoden der enzymatischen Analyse, pp. 1634-1639. Weinheim: Verlag Chemie 1970
Acknowled,~ements. The investigation was supported by grants from NOVO Research Foundation and Idraettens Forskningsr~td. Lisbeth Kall, Vibeke Ulrik and Rikke Gr~nholt performed skilIed technical assistance.
REFERENCES 1. Albano, J. D. M., Ekins, R. P., Maritz, G., Turner, R. C.: A sensitive, precise radioimmunoassay of serum insulin relying on charcoal separation of bound and free hormone moieties. Acta endocr. (Kbh.) 70, 487--509 (1972) 2. Baldwin, K. M., Reitman, J. S., Terjung, R. L., Winder, W. W., Holloszy, J. O.: Substrate depletion in different types of muscle and in liver during prolonged running. Amer. J. Physiol. 225, 1045-1050 (1973) 3. Bellinger, L. L., Mendel, V. E. : Hormone and glucose responses to serial cardiac puncture in rats. Proc. Soc. exp. Biol. (N. Y.) 148, 5 - 8 (1975) 4. Christensen, N. J.: Plasma noradrenaline and adrenaline in patients with thyrotoxicosis and myxedema. Clin. Sci. 45, 163-171 (1973) 5. Claus, T. H., Pilkis, S. J.: Regulation by insulin of gluconeogenesis in isolated rat hepatocytes. Biochim. biophys. Acta (Amst.) 421, 246-262 (1976)
Received September 9, 1976
Pflfigers Archiv
25 (1977)
European Journal of Physiology 9 by Springer-Verlag 1977
Lack of Influence of Glucagon on Glucose Homeostasis after Prolonged Exercise in Rats H. GALBO, E. A. RICHTER, J. J. HOLST, and N. J. CHRISTENSEN Institute of Medical Physiology B, University of Copenhagen, DK-2100 Copenhagen, Department of Clinical Chemistry, Bispebjerg Hospital, DK-2400 Copenhagen Second Clinic of Internal Medicine, Kommunehospitalet, DK-8000, •rhus, Denmark
Summary. The significance of glucagon for postexercise glucose homeostasis has been studied in rats fasted overnight. Immediately after exhaustive swimming either rabbit-antiglucagon serum or normal rabbit serum was injected by cardiac puncture. Cardiac blood and samples of liver and muscle tissue were collected before exercise and repeatedly during a 120 min recovery period after exercise. During the post-exercise period plasma glucagon concentrations decreased but remained above pre-exercise values in rats treated with normal serum, while rats treated with antiglucagon serum had excess antibody in plasma throughout. Nevertheless, all other parameters measured showed similar changes in the two groups. Thus after exercise the grossly diminished hepatic glycogen concentrations remained constant, while the decreased blood glucose concentrations were partially restored. Simultaneously concentrations in blood and serum of the main gluconeogenic substrates, lactate, pyruvate, alanine and glycerol declined markedly. During the post-exercise period NEFA concentrations in serum and plasma insulin concentrations remained increased and decreased, respectively, while plasma catecholamines did not differ from basal values. Muscle glycogen concentrations decreased slightly. These findings suggest that in the recovery period after exhaustive exercise the increased glucagon concentrations in plasma do not influence gluconeogenesis.
known to imply activation of gluconeogenesis [5, 7, 17, 19]. Since it is clear from many experiments with preparations of rat liver that glucagon can stimulate gluconeogenesis strongly [ 5 - 7 , 17] this leads to the general supposition that such an effect occurs in vivo. However, definitive experiments with regard to the role of glucagon in control of gluconeogenesis in vivo have not yet been carried out. In the present study antiglucagon antibodies were administered to rats immediately after exhaustive swimming in order to evaluate the influence of endogenous pancreatic glucagon on glucose homeostasis in the post-exercise period.
MATERIALS AND METHODS
INTRODUCTION
51 male, 10-- 12 h fasted Wistar rats were used in the experiments. Eight rats were resting controls, while 43 rats weighing 2 5 0 - 270 g were forced to swim with a tail weight (4 ~ of body weight) in water maintained at 3 3 - 34~C until exhaustion. At termination of exercise, the animals were immediately anesthetized with ether. 10 rats were then sacrificed, while in 33 rats 0.2 ml of blood were drawn by cardiac puncture for glucose analysis. Then 0.5 ml of either normal rabbit serum (N-rats, n -- 18) or rabbit-antiglucagonserum (A-rats, n = 15) were injected through the cardiac cannula and the rats were placed in cages with access to water, but not to food. 30 rain after exercise 9 N-rats and 7 A-rats were sacrificed. 120 rain after exercise 9 N-rats and 8 A-rats were sacrificed. 30 and 60 min post-exercise these rats were superficially anesthetized with ether and had 0.2 ml of blood drawn by cardiac puncture for glucose analysis. At sacrifice all animals were anesthetized with ether and 6 - 8 ml of blood (A- and N-rats, respectively) were drawn into heparinized syringes for analysis of hormones and substrates 1. Samples of the liver, of the superficial part of the vastus lateralis muscle (which consists predominantly of fast-twitch white fibers [2]), of the deep portion of the vastus lateralis muscle (predominantly fast-twitch red fibers [2]), and of the soleus muscle (predominantly slow-twitch red fibers [2]) were quickly removed and frozen in liquid nitrogen.
Glucagon secretion is enhanced during brief fasting [7, 17], certain forms of diabetes [7, 17], and during the recovery period after exercise [10], conditions
1 Serial cardiac puncture and ether anesthesia has previously been shown to increase plasma glucose concentrations, whereas insulin was not changed [3]
Key words." Insulin -
Epinephrine phrine - Glycogen - Gluconeogenesis.
Norepine-
22
Pflfigers Arch. 369 (1977)
Table 1. The influence on liver and muscle glycogen (mmoles of glucose/kg of wet tissue) of exercise and of administration immediately after exercise of antiglucagon-antiserum Before exercise
0 min postexercise
30 rain postexercise
120 min postexercise
Liver
354 _+ 26 (8)
12 i 2 + (10)
A-rats N-rats
13 • i + 16 • 3 +
(7) (9)
11 • i + 12 4- 1 +
Superficialvastus
33 _+ 2
(8)
17 + 2 + (10)
A-rats N-rats
14 • 2 + 14 • 3 +
(6) (8)
11 • 2 § (8) 8 4- 2 +* (8)
Deepvastus
23
_+ 2
(8)
12 4- 2 + (10)
A-rats N-rats
11 + 1 + 12 + 2 +
(7) (9)
10 • 3 + 8 • 1+
(7) (9)
Soleus
18
4- 1
(8)
18 4- 2
A-rats N-rats
14 • 1 + (7) 12 • I +* (9)
13 • 2 + 14 • I +
(8) (9)
(9)
(7) (9)
Values are mean +_ S.E. N u m b e r of observations are shown in parenthesis. A- and N-rats were treated with rabbit antiglucagon-antiserum or normal rabbit serum, respectively. + and * denote that values are significantly different from values before exercise and values immediately postexercise (0 min postexercise), respectively
Blood pyruvate was determined by an enzymatic fluorimetric method [16], and plasma alanine was determined by the alan• dehydrogenase method [20]. Epinephrine and norepinephrine were determined by a previously published radio-enzymatic assay [4] modified for m e a s u r e m e n t of very small amounts. Insulin was determined by radioimmunoassay [1]. The detection limit for the assay system was 0.15 pmol/1. The methods of sampling and analysis of blood and tissue for glucagon, antiglucagon-antibody, glucose, lactate, N E F A (non-ester• fatty acids), glycerol, and glycogen have been reported elsewhere [9]. All analyses of a h o r m o n e were carried out in a single assay run. The characteristics of the antiglucagon-serum, which was administered to the rats, has been described in detail elsewhere [13]. The statistical evaluation of the data was made by t-test using the test for paired data when applicable. Differences of m e a n s were considered statistically significant when P < 0.05.
RESULTS
GLUCOSE mmol I [ 6 5
[ SWIMMING
I
Injection of i, Antig(ucagon t antiserum
k
I Norm~ rabbit serum
/, 3 2
I
I
-90
0
GLUCAGON prnol / I 700
I
I
30
60
I 120
i I i
T
500
The Exercise Period. The duration of swimming was
92 • 3 (S.E.) rain (n = 43), and did not differ significantly between rats sacrificed immediately, 30 or 120 min after exercise. During exercise a marked diminution of the hepatic glycogen content (Table 1) was accompanied by a decline in blood glucose concentrations (Fig. 1) which was identical in the various groups of swimming rats. Simultaneously, the glycogen concentrations in the deep and superficial part of the vastus muscle were halved (Table 1). The concentrations of NEFA and glycerol in serum and of lactic acid in blood increased, while the concentrations of pyruvate and alanine in plasma did not change significantly (Fig. 2). During exercise the plasma concentrations of glucagon increased 8-fold, while plasma concentrations of insulin decreased dramatically (Fig. 1). Immediately after exercise, glucose concentrations correlated significantly with glucagon concentrations (r = - 0.96, P < 0.001), but not with insulin concentrations.
300 100 I
I
-90
0
I
1
30
120
INSULINI
pmol / I 60
20
i \
5
I - 90
,i . . . .
~ 1 =::.1 0
30
rnin
. . . . =----!I 120
postexercise
Fig. 1. The concentrations of glucose in blood and of glucagon and insulin in plasma during and after exhaustive exercise. (7 pmol 9 1-1 of insulin = 1 g U - m l - 9
H. Galbo et al. : Glucagon and Post Exercise Glucose Homeostasis LACTATE mmol / I
GLYCEROL mmol / l
[ SWIMMI[~ I
0.4
? Antiglucagon ] ant iserurn ~ or Normal rabbit"J serum* I
-90
"~ 0.2 J
30
-90
I
1
0
30
I
120
NEFA meq / !. 1,6
t'-,,
1.2
too ,,
I
i I
-90
l
120
i
5o
J i
+
J
0
.I
0.3
PYRUVATE jJmol/[ 15o
23
0
[
9
30
0.8 0.4
120
t !
-90
600 t ALAN]NE [ pmo[ / l
I
I
|
0
30
120
min
postexercise
400 ~,
-.'.
-.-,.l
1
200
J
i I
-90
&
1
30
The Post-Exercise Period. After exercise, except for plasma glucagon concentrations, the course of the different variables was identical whether antiglucagonserum or normal serum had been administered (Figs. 1 and 2, Tables 1 and 2). Throughout the post-exercise period the hepatic glycogen concentrations remained at the low levels reached at exhaustion while muscle glycogen concentrations tended to decrease further (Table 1). Blood glucose concentrations initially increased but from 30 to 120 rain post-exercise blood glucose concentrations remained constant 20 % below pre-exercise values. While in the postexercise period blood glucose concentrations thus were partially restored the concentrations of the gluconeogenic substrates lactate, pyruvate, alanine, and glycerol gradually declined (Fig. 2). 30, 120, and 120 rain after exercise, respectively, the concentrations of pyruvate and lactate in blood and of alanine in plasma were significantly below pre-exercise levels. 120 rain after exercise the concentrations of glycerol in serum no longer significantly exceeded pre-exercise
Fig. 2. The concentrations of lactate and pyruvate in blood, of alanine in plasma, and of glycerol and N E F A in serum during and after exhaustive exercise
Table 2. The plasma concentrations of epinephrine and norepinephrine in rats before exercise and 30 rain after exercise. Exercised rats were treated with either rabbit antiglucagon-antiserum (A-rats) or normal rabbit serum (N-rats) immediately after exercise. Values are mean 4- S.E. Number of observations are shown in parenthesis Before exercise
30 rnin postexercise A-rats
N-rats
Epinephrine (ng/ml)
0.46 4- 0.10 (4)
0.93 _+ 0.35 (5)
0.96 4- 0.33 (4)
Norepinephrine (ng/ml)
0.93 4- 0.33 (4)
1.12 _+ 0.30 (5)
0.72 4- 0.15 (5)
values, whereas NEFA concentrations still were markedly increased (Fig. 2). Thirty minutes after exercise the plasma concentrations of epinephrine and norepinephrine did not differ significantly from concentrations at rest
24 (Table 2). Insulin concentrations in plasma, however, initially increased slightly (P < 0.01), but stayed far below pre-exercise values throughout the post-exercise period (Fig. 1). In rats treated with normal serum the glucagon concentrations in plasma decreased markedly during the first 30 min of the post-exercise period but then glucagon concentrations remained constant at about twice the pre-exercise values (Fig. 1). All rats treated with antiglucagon-serum had "excess" antibody [9, 13] in plasma at sacrifice. The "titer" [9, 13] varied from 1:500 to 1:2000.
DISCUSSION In the present study of the recovery phase after exercise the grossly diminished hepatic glycogen content was constant. Accordingly after exercise the glucose necessary not only for the metabolism of the obligatory glycolytic tissues but also for the partial restoration of extracellular glucose concentrations was produced entirely by gluconeogenesis. At rest in the post absorptive state, however, only a minor part (25 ~) of hepatic glucose production is derived from gluconeogenesis [19]. In addition, the major non obligatory glycolytic tissues (muscle [19] and adipose tissue) only consume small amounts of glucose in the basal state. Thus, even though decreased glucose uptake in non obligatory glycolytic tissues in the post-exercise period-however in contrast to the previous finding of increased muscular glucose uptake after exercise [19]-can not be excluded due to lack of direct flux determinations, available evidence strongly suggests that in the present study gluconeogenesis after exercise was increased compared with the preexercise period. While gluconeogenesis probably increased after exercise, the concentrations of lactate and pyruvate in blood and of alanine in plasma declined below preexercise levels supporting previous findings [19] of increased fractional extraction of these main gluconeogenic substrates [7] in the post-exercise period. Since glucagon concentrations in plasma remain elevated above basal levels in the immediate postexercise period (Fig. 1) [10] increased glucagon secretion has been suggested to be of importance for the accellerated rate of hepatic gluconeogenesis in the recovery phase after exercise [19]. We have recently shown that administration of antiglucagonserum to rats ensures a rapid and extensive neutralization of endogenous glucagon during a period of time comparable to the post-exercise period in the present study [13]. Nevertheless, in the present study, after exercise tissue glycogen and blood glucose concentrations were identical whether antiglucagon-serum
Pfliigers Arch. 369 (1977) or normal serum had been administered. The lack of difference in gluconeogenesis between rats treated with antiglucagon-serum and rats treated with normal serum can neither be explained by differences in the hepatic supply of essential substrates (Fig. 2) [7], nor by differences in the plasma concentrations of insulin (Fig. 1) and catecholamines (Table 2), hormones which might rapidly influence the rate of gluconeogenesis [5-7, 17]. It appears from these results that the increased concentrations of glucagon in plasma do not significantly influence hepatic glucose production in the post-exercise period, a finding which contrasts with the previously reported observation that during exercise neutralization of glucagon diminishes hepatic glucose production [9]. The discrepancy may be explained by larger glucagon concentrations during than after exercise (Fig. 1), by larger sensitivity of glycogenolysis than of gluconeogenesis to stimulation by glucagon [17] and by larger binding of glucagon and larger glucagon-stimulated cyclic AMP accumulation and glucagon-stimulated glucose production in fed than in glycogen deprived hepatic cells [5,6,8]. Furthermore, since the hyperglycemic effect of glucagon is highly dependent upon insulinopenia [12, 17, 18] the somewhat larger insulin concentrations after than during exercise (Fig. 1) possibly minimized the influence of glucagon on carbohydrate metabolism. At present we have to propose that induction during exercise ofgluconeogenic enzymes [7] possibly brought about by increased release of lactate [7], glucocorticolds [7, 14, 15], and glucagon [17] partly accounts for the increased rate of gluconeogenesis in the recovery phase after exercise. Sometimes increased hepatic supply of substrates in the post-exercise period [19] may contribute to the increased glucose formation [7], and sometimes low insulin concentrations may do so [6, 7]. After non-exhaustive exercise increased concentrations of insulin as well as of glucose have been found [19], but after exhaustive exercise in the present study the concentrations of these substances were below basal levels (Fig. 1). When glucose concentrations 30 min after exercise approached basal levels, glucagon concentrations simultaneously declined markedly while insulin concentrations remained almost constant (Fig. 1), possibly reflecting the fact that at low glucose levels the pancreatic alpha cell is more sensitive than the beta cell to changes in glucose concentrations [11]. The finding of similar rates of gluconeogenesis and of similar plasma substrate concentrations (Fig. 2) in rats treated with antiglucagon-serum compared to rats treated with normal serum indicates that glucagon does not influence peripheral metabolism in the postexercise period. Thus it appears that glucagon is not indispensable for the mobilization of free fatty acids
H. Galbo et al. : Glucagon and Post-Exercise Glucose Homeostasis
25
neither after exercise nor, as shown previously [9,15] during exercise. Low insulin concentrations (Fig. 1), on the other hand, may have decreased the uptake and oxidation of glucose in the muscles and may accordingly have contributed to the low concentrations in blood and serum of pyruvate, lactate and alanine [19]. Furthermore, the low insulin concentrations probably promoted lipolysis and also thereby served to preserve blood glucose concentrations in spite of a probably increased total metabolic rate [10] in the recovery period after exercise. The present study has shown that in rats fasting during recovery after prolonged exhausting exercise, extracellular glucose concentrations are partially restored even though hepatic glycogen concentrations are "not further diminished. The increased glucagon concentrations in plasma found during the postexercise period do not significantly influence glucose production in this period.
6. Davidson, M. J., Bertiner, J. A. : Acute effects of insulin on carbohydrate metabolism in rat liver slices: independence from glucagon. Amer. J. Physiol. 227, 7 9 - 8 7 (1974) 7. Exton, J. H. : Gluconeogenesis. Metabolism 21, 945 - 990 (1972) 8. Fouchereau-Peron, M., Rancon, F., Freychet, P., Rosselin, G. : Effect of feeding and fasting on the early steps of glucagon action in isolated rat liver cells. Endocrinology 98, 755-760 (1976) 9. Galbo, H., Holst, J. J.: The influence of glucagon on hepatic glycogen mobilization in exercising rats. Pflfigers Arch. 363, 4 9 - 53 (19761 10. Galbo, H , Holst, J.J., Christensen, N.J., Hilsted, J.: Glucagon and plasma catecholamines during beta-receptor blockade in exercising man. J. appl. Physiol. 40, 855-863 (1976) 11. Gerich, J. E., Charles, M. A., Grodsky, G. M.: Characterization of the effects of arginine and glucose on glucagon and insulin release from the perfused rat pancreas. J. clin. Invest. 54, 833-841 (1974) 12. Gerich, J. E., Lorenzi, M,, Bier, D. M., Tsalikian, E., Schneider, V., Karam, J. H., Forsham, P. H. : Effects of physiologic levels of glucagon and growth hormone on human carbohydrate and lipid metabolism. J. clin. Invest. 57, 875-884 (1974) 13. Holst, J. J., Galbo, H., Richter, E. A.: Neutralization of glucagon by antibodies. (To be submitted) 14. Huibregtse, C. A., Brunsvold, R. A., Ray, P. D.: Dietary and hormonal regulation of some enzyme activities associated with gluconeogenesis in rabbit liver. Biochim. biophys. Acta (Amst.) 421, 228 - 236 (1976) 15. Luyckx, A. S., Dresse, A., Cession-Fossion, A., Lefebvre, P. J. : Catecholamines and exercise-induced glucagon and fatty acid mobilization in the rat. Amer. J. Physiol. 229, 376-383 (1975) 16. Olsen, C.: An enzymatic fluorimetric micromethod for the determination of acetoacetate, fl-hydroxybutyrate, pyruvate and lactate. Clin. chim. Acta 33, 293-300 (1971) 17. Park, C. R., Exton, J. H.: Glucagon and the metabolism of glucose. In: Glucagon (P. J. Lefebvre and R. H. Unger, eds.) Oxford: Pergamon Press 1972 18. Sherwin, R. S., Fisher, M., Hendler, R., Felig, P.: Hyperglucagonemia and blood glucose regulation in normal, obese and diabetic subjects. New Engl. J. Med. 294, 455-461 (1976) 19. Wahren, J., Felig, P., Hendler, R., Ahlborg, G.: Glucose and amino acid metabolism during recovery after exercise. J. appl. Physiol. 34, 838-845 (1973) 20. Williamson, D. H.: Methoden der enzymatischen Analyse, pp. 1634-1639. Weinheim: Verlag Chemie 1970
Acknowled,~ements. The investigation was supported by grants from NOVO Research Foundation and Idraettens Forskningsr~td. Lisbeth Kall, Vibeke Ulrik and Rikke Gr~nholt performed skilIed technical assistance.
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Received September 9, 1976
