Effect of low blood glucose on plasma CRF, ACTH, and cortisol during prolonged physical exercise IZUMI TABATA, FUTOSHI OGITA, MOTOHIKO Department of Physiology and Biomechanics, National Kagoshima Prefecture 891-23, Japan TABATA, IZUMI, FUTOSHI OGITA, MOTOHIKO MIYACHI, AND HIDETARO SHIBAYAMA. Effect of low blood glucose on plasma CRF, ACTH, and cortisol during prolonged physical exercise. J.
Appl. Physiol. 71(5): 1807-1812, 1991.-The effects of low blood glucose concentration during low-intensity prolonged physical exercise on the hypothalamus-pituitary-adrenocortical axis were investigated in healthy young men. In experiment 1, six subjectswho had fasted for 14 h performed bicycle exerciseat 50% of their maximal 0, uptake until exhaustion. At the end of the exercise,adrenocorticotropic hormone (ACTH) and cortisol increased significantly. However, this hormonal responsewas totally abolishedwhen the samesubjectsexercised at the sameintensity while blood glucoseconcentrations were maintained at the preexerciselevel. In experiment 2, in addition to ACTH and cortisol, the possiblechangesin plasmaconcentration of corticotropin-releasing factor (CRF) were investigated during exercise of the sameintensity performed by six subjects.As suggestedby a previous study (Tabata et al. Clin. Physiol. Oxf. 4: 299-307,1984), when the blood glucoseconcentrations decreasedto ~3.3 mM, plasmaconcentrations of CRF, ACTH, and cortisol showeda significant increase.At exhaustion, further increaseswere observedin plasma CRF, ACTH, and cortisol concentrations. These results demonstrate that decreasesin blood glucoseconcentration trigger the pituitaryadrenocortical axis to enhancesecretion of ACTH and cortisol during low-intensity prolonged exercise in humans. The data alsomight suggestthat this activation is due to increasedconcentration of CRF, which was shown to increase when blood glucoseconcentration decreasedto a critical level of 3.3 mM. human; hypothalamus-pituitary-adrenocortical axis
MIYACHI, AND HIDETARO SHIBAYAMA Institute of Fitness and Sports, Kanoya City,
load may have modulated ACTH release by mechanisms other than counteracting low circulating glucose concentrations. Therefore, to confirm the relationship between blood glucose concentrations and ACTH and cortisol release, we decided to investigate the effects of maintaining normal blood glucose concentrations on plasma ACTH and cortisol concentrations during exercise. This method has advantages for confirming the causal relationship between low blood glucose and plasma ACTH and cortisol responses during prolonged exercise. Because circulating glucose levels can be sufficiently clamped at the basal level, the role of the fall in circulating glucose in the control of hormone release was isolated from other humoral factors that may change and possibly affect the HPA axis during long-term physical exercise. A new technique recently became available to measure plasma concentrations of corticotropin-releasing factor (CRF), which is secreted from the hypothalamus and may stimulate ACTH secretion from the pituitary. We thought it would be of interest to use this technique to examine the responses of the HPA axis to low blood glucose concentrations, especially a blood glucose level of 3.3 mM, because previous studies (l&16) suggested that the HPA axis is stimulated during low-intensity prolonged physical exercise in humans. MATERIALS
AND METHODS
The study consisted of two experiments. Subjects
that serum adrenocorticotropit hormone (ACTH) and cortisol concen tratio ns increase during prolonged physical exercise at a low intensity [ -50% maximal 0, uptake (vo2 m,)] (X,16). On the basis of the results of this experiment, we suggested that the central stimulus, which may be generated in the hypothalamic region after the decreased blood glucose concentration has been sensed , may stimulate the hypothalamus-pituitary-adrenocortical (HPA) axis and induce ACTH secretion during this kind of exercise. We demonstrated that high concentrations of ACTH and cortisol in blood at the exhaus tion point of low- intensity prolonged exerci se in h umans were depressed a.fter the subjects ingested a glucose solution and blood glucose levels rapidly increased to normal. However, it is not possible to conclude that the decrease in blood glucose is the only factor to trigger ACTH release during low-intensity prolonged exercise in humans, because ingestion of a major glucose WE HAVE DEMONSTRATED
0161-7567/91 $1.50 Copyright
0
Six physical education students ,20-25 yr of age, volunteered for the study. After a deta iled explanation of the purpose, potential benefits, and risks associated with participation, the students provided written informed consents. Their physical characteristics are shown in Table 1. Protocol Experiment la. The subjects of experiment 1 exercised twice, without and with glucose infusion (experiments la and 1 b, respectively), and ate the same eucaloric meals for 3 days before exercises. On the day of experiment la, the subjects reported to the laboratory at 0800, after a 14-h overnight fast. A polyethylene catheter was inserted percutaneously into an antecubital vein for blood sampling. A thermocouple was inserted 15 cm beyond the anus to measure core temperature. The subjects rested
1991 the American
Physiological
Society
1807
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1808 TABLE
HPA
No.
1 2 Values
TO
LOW
BLOOD
n
Yr
Weight, kg
6 6
21+1 23kl
65.Ok6.7 63.923.3.
are means
+ SD. \jo2 max, maximal
VO
ml
l
kg-’
2 maw l
GLUCOSE
IN
PHYSICAL
EXERCISE
Methods . vo 2 m8Xwas determined
1. Characteristics of the subjects Age,
Expt
RESPONSES
min-’
61.2k3.4 53.1k6.2
0, uptake.
for 30 min, after which expired gas was collected for 10 min and blood was sampled. At -0900, the subjects began to exercise on a bicycle ergometer (Monark, Sweden) at a load that required -50% vo2 max. The exercise was continued until the subjects became exhausted. Blood was taken every 10 min during the exercise and immediately before the end of the exercise. During the exercise, expired gas was collected in a Douglas bag for 1 min every 10 min. Heart rate (HR), rectal temperature (T,,), and rating of perceived exertion (RPE) were recorded every 10 min during exercise. During exercise in experiment la, no infusion was given. Experiment 1b. Two weeks after experiment la, the same subjects participated in experiment lb. The protocol was identical to that of experiment la, except another catheter was inserted into the antecubital vein of the other arm for infusion of 20% glucose solution (Otsuka, Japan). Blood was sampled every 10 min and analyzed immediately. The blood glucose level was maintained at the value obtained before exercise by use of an infusion pump (model STC 521, Terumo, Japan). Because the previous study (15, 16) demonstrated that blood glucose concentration did not change until exercise time exceeded 60 min, the glucose infusion started 60 min after initiation of the exercise unless the blood glucose concentration had decreased by 0.3 mM from the preexercise value. To determine the initial infusion rate to maintain the blood glucose level at the preexercise value, pilot experiments were performed to find individual infusion rates by a trial-and-error method. In experiment lb, several additional blood samples (4 ml) were drawn between the lo-min intervals to measure current blood glucose concentration and to adjust the infusion rate minutely by a negative-feedback principle. After the initial infusion period, the infusion rate was increased as exercise continued. The actual infusion rate was 0.26 t 0.10 (SD) g/min from 60 min of exercise to 0.57 t 0.14 g/min during the last infusion period. With this method, observed blood glucose concentrations never differed from the preexercise level by >0.5 mM. Exercise was continued for the same duration as in experiment la. After exercise was completed, the infusion was terminated. Experiment 2. To study whether the plasma ACTH and cortisol increase during prolonged physical exercise is triggered by CRF from the hypothalamus, plasma CRF concentrations were measured in blood samples drawn from subjects who exercised according to the same protocol as experiment la. Furthermore, for the specific purpose of determining whether plasma CRF concentration increases simultaneously with plasma ACTH and cortisol, additional blood was sampled when blood glucose concentration had decreased to ~3.3 mM, which had been reported to be the critical level for triggering ACTH secretion during prolonged exercise (15, 16).
by the leveling-off criterion (6, 17). After a linear relationship had been determined for each subject between exercise intensity (watt) and the steady-state 0, uptake measured during the last 2 min of a lo-min constant submaximal-intensity exercise bout (6-9 points), 0, uptake was measured for the last two 30-s intervals during several bouts of supramaximal-intensity exercise that lasted 2-8 min. After leveling-off of 0, uptake to the increase in exercise intensity was visually confirmed, it was considered as a VO~,~, of the subjects. Measurement of V02max normally took 2-3 days. These preliminary exercise tests were done at least 4 days before the experiments. Expired gas volume was measured with a gasometer (Shinagawa Seisakusho, Japan). The concentration of 0, and CO, was analyzed using a mass spectrometer (MGA1100, Perkin-Elmer). Blood glucose and lactate concentrations were measured using an automatic glucose analyzer (YSI 23A, Yellow Springs Instrument, Yellow Springs, OH). Plasma CRF, ACTH, and cortisol concentrations were determined by radioimmunoassay in duplicate. The intra-assay coefficients of variation of CRF, ACTH, and cortisol were 21.4% (mean 4.9 rig/l, n = 6), 2.0% (mean 121 rig/l, n = lo), and 4.7% (mean 108 PgIl, n = lo), respectively. The interassay coefficients of variation of CRF, ACTH, and cortisol were 14.3% (mean 7.4 rig/l, n = 5), 5.0% (mean 116 rig/l, n = lo), and 6.5% (mean 101 pgll, n = 5), respectively. The assay sensitivities of CRF, ACTH, and cortisol were 3.0 rig/l, 2 rig/l, and 3 pgll, respectively. Cross-reactivity of ACTH to ,&endorphin, ,&lipotropin, and a-melanocyte-stimulating hormone was not detectable up to a concentration of lo2 ,ugll. Cross-reactivity of cortisol to prednisolone was 60-70%. Blood samples were drawn every 10 min during exercise. However, because of the high cost of the hormone assay, concentrations of the three hormones were measured from samples taken before exercise, every 30 min during exercise, and at the end of the exercise. In addition, in experiment 2, hormone concentrations were determined for the sample that was drawn when blood glucose concentration decreased to 0.10). Plasma cortisol concentrations also did not change during exercise (P > 0.10).
As a result, plasma ACTH and cortisol concentrations did not differ between the values with and without glucose infusion up to 90 min of exercise. However, at the end of exercise with glucose infusion, plasma ACTH and cortisol concentrations were significantly lower than those without glucose infusion (P < 0.01 and P < 0.001, respectively). Experiment 2. Exercise intensity was 113 t 15 W. Six subjects performed this low-intensity exercise for 153 t 46 min (60-210 min). 0, uptake during exercise was 1.71 t 0.13 Vmin, which elicited 50.6 t 2.6% VO, maxat 30 min of exercise. Thereafter, 0, uptake tended to increase. However, it never exceed 60% VO, max during the entire exercise period (Table 3). Blood glucose concentrations decreased to ~2.7 mM at the end of exercise (Fig. 2). Plasma CRF concentrations did not change significantly for the 60 min of exercise. A significant increase of this hormone was observed (P < 0.001) when blood glucose concentrations became ~3.3 mM (128 t 47 min). Further elevation of this hormone was observed toward the end of the exercise period (Fig. 2). Plasma ACTH and cortisol concentrations followed the change in the plasma CRF concentration during the exercise (Fig. 2). T,, increased during exercise but was not >38.3”C (Table 3). DISCUSSION
The stimulus that provoked hormone secretion during the later phase of exercise in experiments la and 2 is not thought to be an exercise-intensity-related factor (2, 3, 8). Because the exercise intensity was ~60% VO, maxand the significant increase of these hormones in the blood was not detected during the earlier phase of exercise, the postulated stimulus is a factor that changed during the later phase of the exercise. As discussed in a previous study, hypoxia (l4), increased body temperature (l), and increased sympathetic activation (4) are not thought to
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1810
HPA \
Q) cn 0 0
5. O-
s-
4 O-
-3E (3
1
. *.
u3s 0
NO
u
0 E
BLOOD 1
GLUCOSE ’
..
.
***
.
Glucose
Glucose
2. 0
0 I
LOW
*. P
,o-o--o
TO f
I
..
E
RESPONSES
’
Infusion
1
I
I
I
60
120
180
240
I
I
60 I
120
I
80
5 cn .m
0 400 ’ t O- --0
SF-
300
c)
W
No
240
Glucose
Glucose
Infusion I,
‘-
K
180
100
1
I
0 60 E xercise
,
I
120 Time
I
1
180 240 (min)
FIG. 1. Effects of maintenance of blood glucose concentrations on plasma ACTH and cortisol concentrations during low-intensity prolonged exercise. Significant differences between values observed during prolonged exercise with or without glucose infusion: **P < 0.01; ***P < 0.001.
be involved in activation of ACTH secretion under the conditions of the present study (15). Galbo et al. (5) demonstrated that the lower the blood glucose concentration, the higher the responses of blood cortisol concentration during prolonged physical exercise. Tabata et al. (15) demonstrated that high concentrations of ACTH and cortisol in blood may be depressed in humans by the ingestion of a glucose solution during low-intensity exercise. Nazar (9) produced the same results in exercising dogs. These results may suggest that, at a certain level, low blood glucose concentrations trigger ACTH release. However, ingestion of a major glucose TABLE
PHYSICAL
EXERCISE
load may have modulated ACTH release by mechanisms other than counteracting low blood glucose concentrations alone. Furthermore it is not clear which glucosesensitive site is most related to the exercise-induced ACTH secretion, because several sites are sensitive to blood glucose concentration (brain and liver). The glucose-receptive site in the liver is affected by high glucose concentrations from the gut, especially after glucose ingestion (10). Consequently, we chose to use an intravenous infusion technique by which the effect of raising blood glucose concentrations should modulate only the brain’s glucose-sensitive site (12). This site is affected only by a decrease in blood glucose concentrations to the lower range. As shown in experiment lb, the plasma ACTH concentration did not change when the blood glucose concentration was maintained by the infusion of glucose. Furthermore we previously showed that an increased concentration of serum ACTH due to prolonged physical exercise of the same type used here was greatly suppressed by increased blood glucose concentrations after glucose ingestion (15). These results strongly suggest that secretion of ACTH by the pituitary is triggered when blood glucose concentrations decrease to a certain level during low-intensity prolonged physical exercise. Wasserman et al. (18) also reported that glucose infusion reduced the response of increased plasma cortisol concentrations induced by a somatostatin infusion-induced decrease in blood glucose concentrations during treadmill exercise in dogs. Widmaier et al. (19) demonstrated that secretion of CRF from the hypothalamus in vitro is regulated by the glucose concentration of the medium. The concentration of glucose that produced no change in the rate of CRF secretion relative to baseline in 5.5 mM glucose was estimated from the dose-response curve to be -3.8 mM. In experiment 2 of the present study, a significant increase in the plasma CRF concentration was observed not only at the end of the exercise period but also when the glucose concentration decreased to ~3.3 mM. This level seems to be the same or a little less than the threshold level of hypothalamus CRF secretion. However, because actual secretion may lag behind, the glycemic sensing threshold may only appear to be lower. This coincidence may indicate that CRF secretion during prolonged physical exercise is triggered in the hypothalamus, which is thought to be influenced by the blood glucose concentration decreasing to the threshold level. Therefore, this activation of the HPA axis may be considered to be the central origin (13). However, the effects of local sensory nervous blockade on plasma CRF, ACTH, and cortisol
3. 0, uptake, HR, T,,, and RPE during prolonged exercise in experiment 2
Before Exercise
0, uptake, l/min HR, beats/min T re9 “C RPE Values
IN
are means
0.245-tO.03
64+8 36.8kO.2
When concn 30 min
60 min
1.71+0.13
1.78+0.22
128+5 37.7kO.5 13.2k1.2
135+7 38.2kO.2 15.0t2.5
blood glucose decreased to ~3.3 mM
1.89IkO.17 143k14 38.1kO.5 18.3k1.8
End
1.84kO.23 152+8 38.2kO.2 20.0
+ SD.
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HPA RESPONSES
2.0 -
I
TO
LOW BLOOD
*** 1
I
1
1
1
0
60
120
180
240
’
1
0
60
120
180
240
0
60
120
180
240
1
I
I
a P
\ ii
I-
h
300
1
I
I
**
-
T
’
0
E xetc
EXERCISE
1811
to-day variation. Therefore we asked the subjects to continue exercise for another 30 min in experiment 1b. Three of the six subjects agreed. No visible changes in hormone concentration were detected. Therefore we believe that inability of adopting randomized order of the two different exercise plans in the present study may not affect the conclusions suggested by the study. In summary, we observed a causal relationship between a significant increase in plasma ACTH and cortisol concentrations and blood glucose concentrations during low-intensity prolonged exercise. These results demonstrate that decreased blood glucose concentrations trigger the pituitary-adrenocortical axis during low-intensity prolonged exercise in humans. The data also suggest that this activation is possibly due to increased concentration of CRF, which was shown to increase when blood glucose concentration decreased to ~3.3 mM, The authors appreciate stimulating discussion with Drs. M. Miyashita and Y. Mutoh (Laboratory for Exercise Physiology and Biomechanics, Faculty of Education, University of Tokyo). Present addresses: F. Ogita, Research Institute of Physical Fitness, Japan Women’s College of Physical Education, 8-19-l Kita-Karasuyama, Setagaya-Ku, Tokyo 157, Japan. M. Miyachi, Kawasaki University of Medical Welfare Science, 288 Matushima, Kurashiki, Okayama 701-01, Japan. Address for reprint requests: I. Tabata, Dept. of Physiology and Biomechanics, National Institute of Fitness and Sports, Shiromizu 1, Kanoya, Kagoshima 891-23, Japan. Received 30 August 1990; accepted in final form 7 July 1991. REFERENCES
2. 3.
I
I
1
60
120
180
ise
IN PHYSICAL
K. J., J. D. FEW, T. J. FORWARD, AND L. A. GIEC. Stimulation of adrenal glucocorticoid secretion in man by raising the body temperature. J. Physiol. Lond. 202: 645-60, 1969. DAVIES, C. T. M., AND J. D. FEW. Effect of exercise on adrenocortical function. J. Appl. Physiol. 35: 887-891, 1973. FARRELI, P. A., T. L. GARTHWAITE, AND A. B. GUSTAFS~N. Plasma adrenocorticotropin and cortisol responses to submaximal and exhaustive exercise. J. AppZ. Physiol. 55: 1441-1444, 1983. FEW, J. D., M. J. GAWEL, F. J. IMMS, AND E. M. TIPTAFL. The influence of the infusion of noradrenaline on plasma cortisol level in man. J. Fhysiol. Lond. 309: 375-389, 1980. GALBO, H., J. J. HOLST, AND H. J. CHRISTENSEN. The effect of different diet and of insulin on the hormonal responses to prolonged exercise. Acta Physiol. Stand. 107: 19-32, 1979. HERMANSEN, L. Oxygen transport during exercise in human subjects. Actu Physiol. Stand. 90, Suppl. 399: l-104, 1974. KJAER, M., N. H. SECHER, F. W. BACH, S. SHEIKH, AND H. GALBO. Hormonal and metabolic responses to exercise in humans: effect of sensory nervous blockade. Am. J. Physiol. 257 (Endocrinol. Metub.
1. COLLINS,
I
:+-A+ 01
GLUCOSE
Tim0
I
4.
240
(mid
FIG. 2. Effects of low-intensity prolonged exercise on blood glucose, plasma corticotropin-releasing factor (CRF), plasma ACTH, and plasma cortisol concentrations. Third point from left indicates mean value observed when blood glucose concentration decreased below 3.3 mM (128 + 47 min). Significant differences from preexercise values: *P < 0.05; **P < 0.01; ***P < 0.001.
concentration must also be studied during this kind of low-intensity prolonged exercise. Using the afferent nervous blockade technique, Kjaer et al. (7) demonstrated that increased ACTH and cortisol concentrations induced by higher-intensity exercise has a local origin (activation of afferent neurons from working muscles). Because it was not possible to predict the exhaustion time for this kind of exercise before the subjects actually exercise, the subjects exercised without glucose infusion first. Thus the method we adopted in the present study was not a randomized procedure. Although we carefully controlled the diet and physical activities of the subjects before the two exercise bouts, there is still a possibility that blood glucose concentration, muscle glycogen content, and overall exhaustion time may be affected by day-
5.
6. 7.
20): E95-ElOl, 8. LUGER, A., MONTGOMERY,
1989.
P. A. DEUSTER, S. B. KYLE, W. T. GALLUCCI, L. C. P. W. GOLD, D. L. LORIA~X, AND G. P. CHROUSOS. Acute hypothalamic-pituitary-adrenal responses to the stress of treadmill exercise. Physiological adaptations to physical training.
N. EngZ. J. Med. 316: 1309-13X1,1987.. 9. NAZAR, K. Adrenocortical activation
during long-term exercise in dogs: evidence for glucostatic mechanism. pfluegers Arch. 329: 156-
166,1977* 10. NIIJIMA,
A. Glucose-sensitive afferent nerve fibres in the hepatic branch of the vagus nerve in the guinea-pig. J. Physiol. Lond. 332:
315-323,1982. 11. ONODERA,
K., AND M. MIYASHITA. A study on Japanese scale for rating of perceived exertion in endurance exercise. Jpn. J. Phys. Educ.
21: 191-203,1976.
OOMURA,~., H. OOYAMA, M. SUGIMORI, T. NAKAMURA, AND Y. YAMADA. Glucose inhibition on the glucose-sensitive neurones in the rat hypothalamus. Nature Lond. 247: 284-286,1974. 13. PLOTSKY, P. M., T. 0. BRUHN, AND W. VALE. Hypophysiotropic 12.
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1812
HPA RESPONSES
TO LOW BLOOD
regulation of adrenocorticotropin secretion in response to insulininduced hypoglycemia. Endocrinology 117: 323-329, 1985. 14. SUTTON, J. R. Effect of acute hypoxia on the hormonal response to exercise. J. Appl. Physiol. 42: 587-592, 1977. 15. TABATA, I., Y. ATOMI, AND M. MIYASHITA. Blood glucose concentration dependent ACTH and cortisol responses to prolonged exercise. Clin. Physiol. Oxf. 4: 299-307, 1984. 16. TABATA, I., Y. ATOMI, Y. MUTOH, AND M. MIYASHITA. Effect of physical training on the responses of serum adrenocorticotropic hormone during prolonged exhaustive exercise. Eur. J. Appl. Physiol. Occup.
Physiol.
61: 188-192,
1990.
GLUCOSE
IN PHYSICAL
EXERCISE
H. L., E. BUSKIRK, AND A. HENSCHEL. Maximal oxygen intake as an objective measure of cardiorespiratory performance.
17. TAYLOR,
J. Appl. Physiol. 8: 73-80, 1955. 18. WASSERMAN, D. H., H. L. A. LICKLEY,
AND M. VRANIC. Interaction between glucagon and other counterregulatory hormones during normoglycemic and hypoglycemic exercise in dogs. J. Clin. Inuest.
74: 1404-1413,1984. 19. WIDMAIER, E. P., P. M. OLOTSKY, S. W. SU~ON, AND W. W. VALVE. Regulation of corticotropin releasing factor secretion in vitro by glucose. Am. J. Physiol. 255 (Endocrinol. Metab. 18): E287E292,1988.
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Appl. Physiol. 71(5): 1807-1812, 1991.-The effects of low blood glucose concentration during low-intensity prolonged physical exercise on the hypothalamus-pituitary-adrenocortical axis were investigated in healthy young men. In experiment 1, six subjectswho had fasted for 14 h performed bicycle exerciseat 50% of their maximal 0, uptake until exhaustion. At the end of the exercise,adrenocorticotropic hormone (ACTH) and cortisol increased significantly. However, this hormonal responsewas totally abolishedwhen the samesubjectsexercised at the sameintensity while blood glucoseconcentrations were maintained at the preexerciselevel. In experiment 2, in addition to ACTH and cortisol, the possiblechangesin plasmaconcentration of corticotropin-releasing factor (CRF) were investigated during exercise of the sameintensity performed by six subjects.As suggestedby a previous study (Tabata et al. Clin. Physiol. Oxf. 4: 299-307,1984), when the blood glucoseconcentrations decreasedto ~3.3 mM, plasmaconcentrations of CRF, ACTH, and cortisol showeda significant increase.At exhaustion, further increaseswere observedin plasma CRF, ACTH, and cortisol concentrations. These results demonstrate that decreasesin blood glucoseconcentration trigger the pituitaryadrenocortical axis to enhancesecretion of ACTH and cortisol during low-intensity prolonged exercise in humans. The data alsomight suggestthat this activation is due to increasedconcentration of CRF, which was shown to increase when blood glucoseconcentration decreasedto a critical level of 3.3 mM. human; hypothalamus-pituitary-adrenocortical axis
MIYACHI, AND HIDETARO SHIBAYAMA Institute of Fitness and Sports, Kanoya City,
load may have modulated ACTH release by mechanisms other than counteracting low circulating glucose concentrations. Therefore, to confirm the relationship between blood glucose concentrations and ACTH and cortisol release, we decided to investigate the effects of maintaining normal blood glucose concentrations on plasma ACTH and cortisol concentrations during exercise. This method has advantages for confirming the causal relationship between low blood glucose and plasma ACTH and cortisol responses during prolonged exercise. Because circulating glucose levels can be sufficiently clamped at the basal level, the role of the fall in circulating glucose in the control of hormone release was isolated from other humoral factors that may change and possibly affect the HPA axis during long-term physical exercise. A new technique recently became available to measure plasma concentrations of corticotropin-releasing factor (CRF), which is secreted from the hypothalamus and may stimulate ACTH secretion from the pituitary. We thought it would be of interest to use this technique to examine the responses of the HPA axis to low blood glucose concentrations, especially a blood glucose level of 3.3 mM, because previous studies (l&16) suggested that the HPA axis is stimulated during low-intensity prolonged physical exercise in humans. MATERIALS
AND METHODS
The study consisted of two experiments. Subjects
that serum adrenocorticotropit hormone (ACTH) and cortisol concen tratio ns increase during prolonged physical exercise at a low intensity [ -50% maximal 0, uptake (vo2 m,)] (X,16). On the basis of the results of this experiment, we suggested that the central stimulus, which may be generated in the hypothalamic region after the decreased blood glucose concentration has been sensed , may stimulate the hypothalamus-pituitary-adrenocortical (HPA) axis and induce ACTH secretion during this kind of exercise. We demonstrated that high concentrations of ACTH and cortisol in blood at the exhaus tion point of low- intensity prolonged exerci se in h umans were depressed a.fter the subjects ingested a glucose solution and blood glucose levels rapidly increased to normal. However, it is not possible to conclude that the decrease in blood glucose is the only factor to trigger ACTH release during low-intensity prolonged exercise in humans, because ingestion of a major glucose WE HAVE DEMONSTRATED
0161-7567/91 $1.50 Copyright
0
Six physical education students ,20-25 yr of age, volunteered for the study. After a deta iled explanation of the purpose, potential benefits, and risks associated with participation, the students provided written informed consents. Their physical characteristics are shown in Table 1. Protocol Experiment la. The subjects of experiment 1 exercised twice, without and with glucose infusion (experiments la and 1 b, respectively), and ate the same eucaloric meals for 3 days before exercises. On the day of experiment la, the subjects reported to the laboratory at 0800, after a 14-h overnight fast. A polyethylene catheter was inserted percutaneously into an antecubital vein for blood sampling. A thermocouple was inserted 15 cm beyond the anus to measure core temperature. The subjects rested
1991 the American
Physiological
Society
1807
Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on November 18, 2018. Copyright © 1991 the American Physiological Society. All rights reserved.
1808 TABLE
HPA
No.
1 2 Values
TO
LOW
BLOOD
n
Yr
Weight, kg
6 6
21+1 23kl
65.Ok6.7 63.923.3.
are means
+ SD. \jo2 max, maximal
VO
ml
l
kg-’
2 maw l
GLUCOSE
IN
PHYSICAL
EXERCISE
Methods . vo 2 m8Xwas determined
1. Characteristics of the subjects Age,
Expt
RESPONSES
min-’
61.2k3.4 53.1k6.2
0, uptake.
for 30 min, after which expired gas was collected for 10 min and blood was sampled. At -0900, the subjects began to exercise on a bicycle ergometer (Monark, Sweden) at a load that required -50% vo2 max. The exercise was continued until the subjects became exhausted. Blood was taken every 10 min during the exercise and immediately before the end of the exercise. During the exercise, expired gas was collected in a Douglas bag for 1 min every 10 min. Heart rate (HR), rectal temperature (T,,), and rating of perceived exertion (RPE) were recorded every 10 min during exercise. During exercise in experiment la, no infusion was given. Experiment 1b. Two weeks after experiment la, the same subjects participated in experiment lb. The protocol was identical to that of experiment la, except another catheter was inserted into the antecubital vein of the other arm for infusion of 20% glucose solution (Otsuka, Japan). Blood was sampled every 10 min and analyzed immediately. The blood glucose level was maintained at the value obtained before exercise by use of an infusion pump (model STC 521, Terumo, Japan). Because the previous study (15, 16) demonstrated that blood glucose concentration did not change until exercise time exceeded 60 min, the glucose infusion started 60 min after initiation of the exercise unless the blood glucose concentration had decreased by 0.3 mM from the preexercise value. To determine the initial infusion rate to maintain the blood glucose level at the preexercise value, pilot experiments were performed to find individual infusion rates by a trial-and-error method. In experiment lb, several additional blood samples (4 ml) were drawn between the lo-min intervals to measure current blood glucose concentration and to adjust the infusion rate minutely by a negative-feedback principle. After the initial infusion period, the infusion rate was increased as exercise continued. The actual infusion rate was 0.26 t 0.10 (SD) g/min from 60 min of exercise to 0.57 t 0.14 g/min during the last infusion period. With this method, observed blood glucose concentrations never differed from the preexercise level by >0.5 mM. Exercise was continued for the same duration as in experiment la. After exercise was completed, the infusion was terminated. Experiment 2. To study whether the plasma ACTH and cortisol increase during prolonged physical exercise is triggered by CRF from the hypothalamus, plasma CRF concentrations were measured in blood samples drawn from subjects who exercised according to the same protocol as experiment la. Furthermore, for the specific purpose of determining whether plasma CRF concentration increases simultaneously with plasma ACTH and cortisol, additional blood was sampled when blood glucose concentration had decreased to ~3.3 mM, which had been reported to be the critical level for triggering ACTH secretion during prolonged exercise (15, 16).
by the leveling-off criterion (6, 17). After a linear relationship had been determined for each subject between exercise intensity (watt) and the steady-state 0, uptake measured during the last 2 min of a lo-min constant submaximal-intensity exercise bout (6-9 points), 0, uptake was measured for the last two 30-s intervals during several bouts of supramaximal-intensity exercise that lasted 2-8 min. After leveling-off of 0, uptake to the increase in exercise intensity was visually confirmed, it was considered as a VO~,~, of the subjects. Measurement of V02max normally took 2-3 days. These preliminary exercise tests were done at least 4 days before the experiments. Expired gas volume was measured with a gasometer (Shinagawa Seisakusho, Japan). The concentration of 0, and CO, was analyzed using a mass spectrometer (MGA1100, Perkin-Elmer). Blood glucose and lactate concentrations were measured using an automatic glucose analyzer (YSI 23A, Yellow Springs Instrument, Yellow Springs, OH). Plasma CRF, ACTH, and cortisol concentrations were determined by radioimmunoassay in duplicate. The intra-assay coefficients of variation of CRF, ACTH, and cortisol were 21.4% (mean 4.9 rig/l, n = 6), 2.0% (mean 121 rig/l, n = lo), and 4.7% (mean 108 PgIl, n = lo), respectively. The interassay coefficients of variation of CRF, ACTH, and cortisol were 14.3% (mean 7.4 rig/l, n = 5), 5.0% (mean 116 rig/l, n = lo), and 6.5% (mean 101 pgll, n = 5), respectively. The assay sensitivities of CRF, ACTH, and cortisol were 3.0 rig/l, 2 rig/l, and 3 pgll, respectively. Cross-reactivity of ACTH to ,&endorphin, ,&lipotropin, and a-melanocyte-stimulating hormone was not detectable up to a concentration of lo2 ,ugll. Cross-reactivity of cortisol to prednisolone was 60-70%. Blood samples were drawn every 10 min during exercise. However, because of the high cost of the hormone assay, concentrations of the three hormones were measured from samples taken before exercise, every 30 min during exercise, and at the end of the exercise. In addition, in experiment 2, hormone concentrations were determined for the sample that was drawn when blood glucose concentration decreased to 0.10). Plasma cortisol concentrations also did not change during exercise (P > 0.10).
As a result, plasma ACTH and cortisol concentrations did not differ between the values with and without glucose infusion up to 90 min of exercise. However, at the end of exercise with glucose infusion, plasma ACTH and cortisol concentrations were significantly lower than those without glucose infusion (P < 0.01 and P < 0.001, respectively). Experiment 2. Exercise intensity was 113 t 15 W. Six subjects performed this low-intensity exercise for 153 t 46 min (60-210 min). 0, uptake during exercise was 1.71 t 0.13 Vmin, which elicited 50.6 t 2.6% VO, maxat 30 min of exercise. Thereafter, 0, uptake tended to increase. However, it never exceed 60% VO, max during the entire exercise period (Table 3). Blood glucose concentrations decreased to ~2.7 mM at the end of exercise (Fig. 2). Plasma CRF concentrations did not change significantly for the 60 min of exercise. A significant increase of this hormone was observed (P < 0.001) when blood glucose concentrations became ~3.3 mM (128 t 47 min). Further elevation of this hormone was observed toward the end of the exercise period (Fig. 2). Plasma ACTH and cortisol concentrations followed the change in the plasma CRF concentration during the exercise (Fig. 2). T,, increased during exercise but was not >38.3”C (Table 3). DISCUSSION
The stimulus that provoked hormone secretion during the later phase of exercise in experiments la and 2 is not thought to be an exercise-intensity-related factor (2, 3, 8). Because the exercise intensity was ~60% VO, maxand the significant increase of these hormones in the blood was not detected during the earlier phase of exercise, the postulated stimulus is a factor that changed during the later phase of the exercise. As discussed in a previous study, hypoxia (l4), increased body temperature (l), and increased sympathetic activation (4) are not thought to
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1810
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be involved in activation of ACTH secretion under the conditions of the present study (15). Galbo et al. (5) demonstrated that the lower the blood glucose concentration, the higher the responses of blood cortisol concentration during prolonged physical exercise. Tabata et al. (15) demonstrated that high concentrations of ACTH and cortisol in blood may be depressed in humans by the ingestion of a glucose solution during low-intensity exercise. Nazar (9) produced the same results in exercising dogs. These results may suggest that, at a certain level, low blood glucose concentrations trigger ACTH release. However, ingestion of a major glucose TABLE
PHYSICAL
EXERCISE
load may have modulated ACTH release by mechanisms other than counteracting low blood glucose concentrations alone. Furthermore it is not clear which glucosesensitive site is most related to the exercise-induced ACTH secretion, because several sites are sensitive to blood glucose concentration (brain and liver). The glucose-receptive site in the liver is affected by high glucose concentrations from the gut, especially after glucose ingestion (10). Consequently, we chose to use an intravenous infusion technique by which the effect of raising blood glucose concentrations should modulate only the brain’s glucose-sensitive site (12). This site is affected only by a decrease in blood glucose concentrations to the lower range. As shown in experiment lb, the plasma ACTH concentration did not change when the blood glucose concentration was maintained by the infusion of glucose. Furthermore we previously showed that an increased concentration of serum ACTH due to prolonged physical exercise of the same type used here was greatly suppressed by increased blood glucose concentrations after glucose ingestion (15). These results strongly suggest that secretion of ACTH by the pituitary is triggered when blood glucose concentrations decrease to a certain level during low-intensity prolonged physical exercise. Wasserman et al. (18) also reported that glucose infusion reduced the response of increased plasma cortisol concentrations induced by a somatostatin infusion-induced decrease in blood glucose concentrations during treadmill exercise in dogs. Widmaier et al. (19) demonstrated that secretion of CRF from the hypothalamus in vitro is regulated by the glucose concentration of the medium. The concentration of glucose that produced no change in the rate of CRF secretion relative to baseline in 5.5 mM glucose was estimated from the dose-response curve to be -3.8 mM. In experiment 2 of the present study, a significant increase in the plasma CRF concentration was observed not only at the end of the exercise period but also when the glucose concentration decreased to ~3.3 mM. This level seems to be the same or a little less than the threshold level of hypothalamus CRF secretion. However, because actual secretion may lag behind, the glycemic sensing threshold may only appear to be lower. This coincidence may indicate that CRF secretion during prolonged physical exercise is triggered in the hypothalamus, which is thought to be influenced by the blood glucose concentration decreasing to the threshold level. Therefore, this activation of the HPA axis may be considered to be the central origin (13). However, the effects of local sensory nervous blockade on plasma CRF, ACTH, and cortisol
3. 0, uptake, HR, T,,, and RPE during prolonged exercise in experiment 2
Before Exercise
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IN
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1.89IkO.17 143k14 38.1kO.5 18.3k1.8
End
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+ SD.
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HPA RESPONSES
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I
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LOW BLOOD
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I
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1
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120
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EXERCISE
1811
to-day variation. Therefore we asked the subjects to continue exercise for another 30 min in experiment 1b. Three of the six subjects agreed. No visible changes in hormone concentration were detected. Therefore we believe that inability of adopting randomized order of the two different exercise plans in the present study may not affect the conclusions suggested by the study. In summary, we observed a causal relationship between a significant increase in plasma ACTH and cortisol concentrations and blood glucose concentrations during low-intensity prolonged exercise. These results demonstrate that decreased blood glucose concentrations trigger the pituitary-adrenocortical axis during low-intensity prolonged exercise in humans. The data also suggest that this activation is possibly due to increased concentration of CRF, which was shown to increase when blood glucose concentration decreased to ~3.3 mM, The authors appreciate stimulating discussion with Drs. M. Miyashita and Y. Mutoh (Laboratory for Exercise Physiology and Biomechanics, Faculty of Education, University of Tokyo). Present addresses: F. Ogita, Research Institute of Physical Fitness, Japan Women’s College of Physical Education, 8-19-l Kita-Karasuyama, Setagaya-Ku, Tokyo 157, Japan. M. Miyachi, Kawasaki University of Medical Welfare Science, 288 Matushima, Kurashiki, Okayama 701-01, Japan. Address for reprint requests: I. Tabata, Dept. of Physiology and Biomechanics, National Institute of Fitness and Sports, Shiromizu 1, Kanoya, Kagoshima 891-23, Japan. Received 30 August 1990; accepted in final form 7 July 1991. REFERENCES
2. 3.
I
I
1
60
120
180
ise
IN PHYSICAL
K. J., J. D. FEW, T. J. FORWARD, AND L. A. GIEC. Stimulation of adrenal glucocorticoid secretion in man by raising the body temperature. J. Physiol. Lond. 202: 645-60, 1969. DAVIES, C. T. M., AND J. D. FEW. Effect of exercise on adrenocortical function. J. Appl. Physiol. 35: 887-891, 1973. FARRELI, P. A., T. L. GARTHWAITE, AND A. B. GUSTAFS~N. Plasma adrenocorticotropin and cortisol responses to submaximal and exhaustive exercise. J. AppZ. Physiol. 55: 1441-1444, 1983. FEW, J. D., M. J. GAWEL, F. J. IMMS, AND E. M. TIPTAFL. The influence of the infusion of noradrenaline on plasma cortisol level in man. J. Fhysiol. Lond. 309: 375-389, 1980. GALBO, H., J. J. HOLST, AND H. J. CHRISTENSEN. The effect of different diet and of insulin on the hormonal responses to prolonged exercise. Acta Physiol. Stand. 107: 19-32, 1979. HERMANSEN, L. Oxygen transport during exercise in human subjects. Actu Physiol. Stand. 90, Suppl. 399: l-104, 1974. KJAER, M., N. H. SECHER, F. W. BACH, S. SHEIKH, AND H. GALBO. Hormonal and metabolic responses to exercise in humans: effect of sensory nervous blockade. Am. J. Physiol. 257 (Endocrinol. Metub.
1. COLLINS,
I
:+-A+ 01
GLUCOSE
Tim0
I
4.
240
(mid
FIG. 2. Effects of low-intensity prolonged exercise on blood glucose, plasma corticotropin-releasing factor (CRF), plasma ACTH, and plasma cortisol concentrations. Third point from left indicates mean value observed when blood glucose concentration decreased below 3.3 mM (128 + 47 min). Significant differences from preexercise values: *P < 0.05; **P < 0.01; ***P < 0.001.
concentration must also be studied during this kind of low-intensity prolonged exercise. Using the afferent nervous blockade technique, Kjaer et al. (7) demonstrated that increased ACTH and cortisol concentrations induced by higher-intensity exercise has a local origin (activation of afferent neurons from working muscles). Because it was not possible to predict the exhaustion time for this kind of exercise before the subjects actually exercise, the subjects exercised without glucose infusion first. Thus the method we adopted in the present study was not a randomized procedure. Although we carefully controlled the diet and physical activities of the subjects before the two exercise bouts, there is still a possibility that blood glucose concentration, muscle glycogen content, and overall exhaustion time may be affected by day-
5.
6. 7.
20): E95-ElOl, 8. LUGER, A., MONTGOMERY,
1989.
P. A. DEUSTER, S. B. KYLE, W. T. GALLUCCI, L. C. P. W. GOLD, D. L. LORIA~X, AND G. P. CHROUSOS. Acute hypothalamic-pituitary-adrenal responses to the stress of treadmill exercise. Physiological adaptations to physical training.
N. EngZ. J. Med. 316: 1309-13X1,1987.. 9. NAZAR, K. Adrenocortical activation
during long-term exercise in dogs: evidence for glucostatic mechanism. pfluegers Arch. 329: 156-
166,1977* 10. NIIJIMA,
A. Glucose-sensitive afferent nerve fibres in the hepatic branch of the vagus nerve in the guinea-pig. J. Physiol. Lond. 332:
315-323,1982. 11. ONODERA,
K., AND M. MIYASHITA. A study on Japanese scale for rating of perceived exertion in endurance exercise. Jpn. J. Phys. Educ.
21: 191-203,1976.
OOMURA,~., H. OOYAMA, M. SUGIMORI, T. NAKAMURA, AND Y. YAMADA. Glucose inhibition on the glucose-sensitive neurones in the rat hypothalamus. Nature Lond. 247: 284-286,1974. 13. PLOTSKY, P. M., T. 0. BRUHN, AND W. VALE. Hypophysiotropic 12.
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1812
HPA RESPONSES
TO LOW BLOOD
regulation of adrenocorticotropin secretion in response to insulininduced hypoglycemia. Endocrinology 117: 323-329, 1985. 14. SUTTON, J. R. Effect of acute hypoxia on the hormonal response to exercise. J. Appl. Physiol. 42: 587-592, 1977. 15. TABATA, I., Y. ATOMI, AND M. MIYASHITA. Blood glucose concentration dependent ACTH and cortisol responses to prolonged exercise. Clin. Physiol. Oxf. 4: 299-307, 1984. 16. TABATA, I., Y. ATOMI, Y. MUTOH, AND M. MIYASHITA. Effect of physical training on the responses of serum adrenocorticotropic hormone during prolonged exhaustive exercise. Eur. J. Appl. Physiol. Occup.
Physiol.
61: 188-192,
1990.
GLUCOSE
IN PHYSICAL
EXERCISE
H. L., E. BUSKIRK, AND A. HENSCHEL. Maximal oxygen intake as an objective measure of cardiorespiratory performance.
17. TAYLOR,
J. Appl. Physiol. 8: 73-80, 1955. 18. WASSERMAN, D. H., H. L. A. LICKLEY,
AND M. VRANIC. Interaction between glucagon and other counterregulatory hormones during normoglycemic and hypoglycemic exercise in dogs. J. Clin. Inuest.
74: 1404-1413,1984. 19. WIDMAIER, E. P., P. M. OLOTSKY, S. W. SU~ON, AND W. W. VALVE. Regulation of corticotropin releasing factor secretion in vitro by glucose. Am. J. Physiol. 255 (Endocrinol. Metab. 18): E287E292,1988.
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