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Département d'Éducation Physique, Université de Montréal, Montreal, Quebec, Canada H3C 3J7
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES
ABSTRACT 
Tadjoré, Maurice, Raynald Bergeron, Martin Latour,
François Désy, Claude Warren, and Jean-Marc Lavoie.
Effects of dietary manipulations and glucose infusion on glucagon
response during exercise in rats. J. Appl.
Physiol. 83(1): 148-152, 1997.
The purpose of the
present investigation was to test the hypothesis that blood glucose
concentration is not always related to glucagon response during
exercise. Three groups of rats were submitted to a prolonged (3-h)
swimming exercise. Two groups of rats had their normal food intake
restricted by 50% the night before the experiment. One of these two
groups of rats was intravenously infused with glucose throughout
exercise to maintain euglycemia. The third group of rats swam while
under normal dietary conditions. Plasma glucose, sampled in arterial
blood, was reduced (P < 0.05) at 75, 105, 150, and 170 min of exercise (from ~130 to 110 mg/dl) in the
food-restricted animals without glucose infusion, whereas a significant
(P < 0.05) increase was measured in
the two other groups during exercise. A significant
(P < 0.01) difference in the mean
integrated areas under the glucose-concentration curve was found only
between the fed and the two food-restricted groups. Plasma insulin
concentrations decreased (P < 0.05)
similarly in all groups during exercise, whereas plasma epinephrine and
norepinephrine concentrations increased significantly
(P < 0.01) in all groups. Despite
differences between groups in plasma glucose response during exercise,
and despite the absence of any decrease in exercising blood glucose
levels in at least two of the three groups, plasma glucagon responses
were increased (P < 0.05) similarly
in all groups (from ~250 to 550 pg/ml) at the end of the exercise
period. The increase in glucagon was significant after 90 min of
exercise in the food-restricted groups, with or without glucose
infusion, but only after 140 min in the fed group. These results
indicate that the glucagon response during exercise is not always
linked to the decrease in plasma glucose.
hepatic glycogen; catecholamines; hypoglycemia
INTRODUCTION IT HAS BEEN REPORTED in numerous investigations that
the liver, through its afferent innervation, is involved in the
regulation of food intake (26, 27). The hepatic afferent
branch of the vagus nerve has also been reported to participate in the
regulation of insulin (17), glucagon (28), and epinephrine (4, 7, 14)
secretion. These effects have been observed under different methodological approaches such as an acute hepatic vagotomy (17, 28),
an insulin-induced hypoglycemia (4, 7, 14), and physical exercise (3,
15). The results of the above-mentioned studies are also in agreement
with some electrophysiological evidence showing a neural link between
the liver and the pancreas and between the liver and the adrenal glands
(22).
The evidence of a link between the liver and the pancreas has brought
us to question the possibility that an hepatic stimulus might influence
the glucagon response during exercise. It is the general view that the
glucagon response is associated with a decrease in plasma glucose.
However, numerous physiological and pathological states such as liver
cirrhosis (13, 20), hyperthyroidism (10), and partial hepatectomy (21)
are characterized by hyperglucagonemia despite the presence of
hyperglycemia or euglycemia. On the contrary, there are hepatic
glycogen-storage disorders such as glycogenoses (absence of
glucose-6-phosphatase) in which normal basal plasma glucagon level is
associated with fasting hypoglycemia (23). These observations have led
Kabadi (11, 12) to suggest that the hyperglucagonemic states are
characterized by a unique metabolic environment, namely hepatic
glycogen depletion.
Similar to the response in resting conditions, plasma glucagon response
during prolonged exercise is often considered as a counterregulatory
response to a decreasing glycemia. This view is supported by the
demonstration that the rise in glucagon is abolished when blood glucose
is increased by glucose ingestion before or during exercise (29).
However, it is possible to find exercise studies where this feedback
mechanism appears to break down. For instance, the glucagon response
during prolonged exercise in dogs (1) and in humans (9) is not affected
by a glucose infusion equivalent to the normal hepatic glucose
production. In a recent study (2), reduced circulating fat in dogs
resulted in a 50% greater glucagon levels during exercise without any
systematic differences in plasma glucose. The purpose of the present
investigation was, therefore, to test the relationship between the
glucagon response and some variations in plasma glucose concentration
during a period of prolonged exercise. Our a priori hypothesis is that blood glucose concentration is not always related to glucagon response during exercise.
METHODS
20 and 0 min) and at different time intervals
during the next 3 h. Collected blood (between 0.075 and 2.0 ml) was
simultaneously replaced with whole blood from an anesthetized donor
animal submitted to the same nutritional conditions as the experimental
animal. The intravenous infusion of either glucose (25% dextrose
solution, mean infusion rate: 1.75 µl/min) or isotonic saline (0.9%)
was made by using a microinfusion pump (model 55-2222, Harvard
Apparatus). Plasma glucose concentrations were measured every 20 min
during the first 2 h and every 10 min during the last hour, and the
infusion rate was adjusted to maintain plasma glucose concentration
>120 mg/dl throughout the exercise session. At the end of the
exercise period, the animals were rapidly taken out of the water and
quickly anesthetized through the arterial catheter by using
pentobarbital sodium (20 mg/kg). Immediately thereafter, the abdominal
cavity was opened and a small piece of liver from the left lobe was
frozen with aluminium block tongs cooled to liquid nitrogen
temperature.
Analytic methods.
Arterial blood was collected into heparinized syringes and separated
into three fractions. The first aliquot of blood (500 µl) was
preserved in Trasylol (50 µl) and centrifuged for 5 min, and the
plasma was stored for glucagon determination. The second fraction of
blood was centrifuged for 5 min (1 min for glucose), and the
supernatant was retained for glucose and insulin analyses. The
remaining part of blood was used for catecholamine determinations; it
was transferred to microtubes containing 50 µl of glutathione (60 mg/ml) and ethylene glycol-bis(
-aminoethyl
ether)-N,N,N
,N-tetraacetic acid (90 mg/ml), kept on crushed ice, and centrifuged immediately for
10 min (5°C; 3,500 revolutions/min). All tissues and blood plasma
were stored at 80°C until analyses were performed.
Plasma glucose concentrations were determined with the use of a glucose
analyzer (model 2300, Yellow Springs Instrument, Yellow Springs, OH).
Insulin and glucagon levels were determined by commercially available
radioimmunoassay kits using porcine insulin and human glucagon
standards, respectively (ICN Biomedicals, Costa Mesa, CA; distributed
by Immunocorp, Montréal, PQ). Catecholamines were
extracted from the plasma according to the procedure described by Remie
and Zaagsma (24) and determined by means of an isocratic high-performance liquid chromatography system (Waters Division, Millipore). The recovery of norepinephrine, epinephrine,
and dihydroxybenzylamine with the concentration of 2 ng/ml was 95.8 ± 8.4, 94.5 ± 4.6, and 79.1 ± 4.3%,
respectively. Liver glycogen content was determined by use
of the phenol-sulfuric acid reaction (18).
Statistical analyses.
All data are reported as means ± SE. The total area under the
concentation curve and above the baseline for glucose was calculated by
using a trapezoidal model. The blood variables were analyzed by a
two-way analysis of variance with repeated-measures design. The Tukey
post hoc test was used in the event of a significant (P < 0.05)
F-ratio. Comparisons of liver glycogen
values and mean areas under the glucose curves were done by using a
one-way analysis of variance.
RESULTS
The hepatic glycogen concentrations measured only at the end of
exercise were 0.77 ± 0.14, 0.3 ± 0.02, and 0.3 ± 0.02 g/100 g for the fed rats and the two food-restricted groups of rats, respectively. These values were significantly
(P < 0.01) higher in the fed than in
the food-restricted rats. Plasma glucose concentrations were
significantly (P < 0.01) reduced at
75, 105, 150, and 170 min of exercise (from ~130 to 110 mg/dl) in the
food-restricted rats without glucose infusion (Fig.
1). The mean total areas under the
glucose-concentration curve for the fed and the food-restricted, with and without glucose infusion, groups were 30,650 ± 1,140 (SE), 26,139 ± 804, and 24,619 ± 795 mg · dl
1 · 180 min1, respectively. A
significant (P < 0.01) difference in
these mean integrated areas was found only between the fed and the two
food-restricted groups. A significant
(P < 0.05) increase in exercising
plasma glucose was observed in the glucose-infused rats. In the fed
rats, plasma glucose concentrations increased significantly
(P < 0.01) throughout the exercise
period (Fig. 1). Plasma insulin concentrations decreased significantly
(P < 0.01) and similarly in all
groups during exercise (Fig. 2). Plasma
epinephrine and norepinephrine concentrations increased significantly
(P < 0.01) with exercise in all
three groups (Fig. 3). No significant
intergroup differences were found for the catecholamine response to
exercise. Glucagon concentrations were, overall, similarly increased
(P < 0.01) in all three groups (from
~250 to 550 pg/ml) at the end of the exercise period (Fig.
4). Glucagon concentrations started to
increase significantly (P < 0.05)
after 90 min of exercise in the food-restricted groups of rats and only
after 140 min in the fed groups of rats (Fig. 4).
, Fast + glucose infusion; 
, fast; 
, fed. * Significantly
different from corresponding resting values,
P < 0.05.
, Fast + glucose infusion; ,
fast; , fed. * Significantly different from all corresponding
exercise values, P < 0.05.
, Fast + glucose infusion; , fast; , fed. * Significantly
different from corresponding resting values,
P < 0.05.
, Fast + glucose infusion; ,
fast; , fed. * Significantly different from corresponding resting values, P < 0.05.
DISCUSSION
It is the general view that the decline in plasma glucose concentrations is the major determinant of the increase in glucagon secretion during prolonged exercise. However, it is possible to find deviations from this general concept in earlier studies (for a review see Ref. 5) as well as in more recent investigations (1, 2, 9). The present study was designed to investigate the possibility that glucagon secretion during exercise is not always related to a decrease in plasma glucose concentration. One of the approaches chosen was to maintain glycemia in food-restricted rats. The results show that, compared with resting values, blood glucose concentrations were significantly (P < 0.05) decreased at 75, 105, 150, and 170 min (Fig. 1) in fasted rats. No such decrease was found throughout exercise in the fasted group of rats infused with glucose or in normally fed rats. Despite these differences in plasma glucose levels, glucagon concentrations were increased significantly at the end of the exercise period in all three groups (Fig. 4). The increase in glucagon during exercise is even more puzzling if one considers that overall blood glucose levels were not decreased during exercise in the food-restricted rats without glucose infusion, as indicated by the areas under the curve. An important distinction in the glucagon response between the groups, however, is the observation that glucagon levels started to increase after 90 min of exercise in both fasted groups of rats, irrespective of the glucose infusion, whereas it started to increase only after 140 min of exercise in the fed group. Overall, these data indicate that an absence of a decrease in blood glucose level during a prolonged exercise period did not prevent glucagon response to be largely increased. In addition, it seems that whether the rats were in the fed or fasted state before exercise influences the time sequence of the subsequent glucagon response. These data, therefore, represent a good indication that a decrease in blood glucose level cannot be the sole determinant of glucagon secretion during exercise.
Similar to the glucagon response, epinephrine and norepinephrine levels were increased in all three groups during exercise. Because these catecholamines were measured only at two time points during exercise, it cannot be known if the time course of these responses was the same for the three groups. There is a tendency for the epinephrine response to be increased more rapidly in the fasted groups of rats without glucose infusion. The catecholamine responses deserve closer consideration because it has been suggested that, in rats, glucagon secretion is mainly due to sympathetic stimulation of alpha-cells (5). This concept, however, is far from being unanimous. For instance, glucose administration in rats has been reported to reduce the exercise-increased glucagon concentrations in some studies (8, 19). It has been reported (5) that the time course of glucagon secretion in exercising rats is similar to that of catecholamine responses. A close look at the data, however, shows that the same relationship of plasma glucagon response could also be established with blood glucose or liver glycogen (30). Contradictory data have led some authors to suggest that both plasma glucose and plasma catecholamines play a major role in exercise-induced glucagon secretion in rats (6). It has also been suggested that in rats unidentified factors are of importance for the glucagon response to exercise (6, 25). In the present study, it cannot be excluded that plasma catecholamines may be associated with the similar increase in glucagon levels measured at the end of exercise. However, both epinephrine and norepinephrine concentrations were significantly elevated after 120 min of exercise in fed rats, which was not the case for glucagon concentrations. It is thus possible that in addition to glucose and/or catecholamines, another factor might be of importance for the glucagon response to exercise.
The reason for using fasted and fed rats in the present study was to
try to draw a parallel between the level of hepatic glycogen content
and the glucagon response during exercise. The results of our study
show that hepatic glycogen values were low in all groups at the end of
exercise, representing at most 10% of normal values in fed conditions
(16). This might constitute a possible explanation for the similar
increase in glucagon in all three groups at the end of exercise. In
addition, the time course of glucagon increases during exercise was
lower in the fed group of rats than in both groups of fasted rats (Fig.
4). It is possible that hyperglycemia in the fed rats might have acted
to prolong the time before the increase in glucagon. However, this
would not explain why glucagon in the same group of rats was sharply increased between 140 and 180 min of exercise while hyperglycemia continued to increase. Although we do not have a time course of the
decrease of liver glycogen during exercise, it is reasonable to assume
that liver glycogen concentrations were higher in the fed group than in
the fasted groups throughout exercise. It is, alternatively, possible
that the slower glucagon response during exercise in the fed rats may
be associated with a higher liver glycogen content. Throughout the
literature, several physiological and pathological states have been
shown to have a distinct relationship between hepatic glycogen content
and plasma glucagon level (for a review, see Ref. 11). Although the
nature of the feedback regulatory mechanism between the liver and the
pancreatic 
-cell is not known, it has been hypothetized that the
hepatic cells secrete a hormonal factor, as a result of high level of
hepatic glycogen content, which would suppress glucagon secretion (11). Conversely, if hepatic glycogen stores are depleted, the secretion of
this substance would be inhibited, resulting in hyperglucagonemia. In
the absence of a clear relationship between plasma glucose and glucagon
response, it is possible that this overall mechanism might contribute
to the increase in glucagon secretion during a period of prolonged
exercise. It is suggested that a new concept relating
liver glycogen content to glucagon might be of importance for the
glucagon response to exercise.
The infusion of glucose during exercise did not have any effect on plasma insulin response compared with the noninfused groups. This is an indication that the quantity of glucose infused was modest. In addition, the low level of liver glycogen at the end of exercise in the infused rats suggests that the glucose infusion did not prevent the remaining liver glycogen from being used during exercise. On the other hand, the increase in blood glucose levels in the fed rats during exercise needs to be addressed. These increases started at the very beginning of the exercise period (20 min) and might reflect a stress component of the swimming exercise. However, because the plasma catecholamine response was not higher in the fed group than in the other groups it cannot be why the glucagon response started to increase only after 140 min of exercise.
In summary, the present study provides evidence that a decrease in blood glucose concentration cannot be the sole determinant of the glucagon response during exercise. It is suggested that a new concept relating liver glycogen content to glucagon might be of importance for the glucagon response to exercise.
ACKNOWLEDGEMENTS
We thank Nathalie Rhéaume for excellent technical assistance.
FOOTNOTES
Address for reprint requests: J.-M. Lavoie, Département d'Éducation Physique, Université de Montrál, C.P. 6128, succ Centre-ville, Montréal, PQ, Canada H3C 3J7 (E-mail: lavoije{at}ere.umontreal.ca).
Received 31 December 1996; accepted in final form 17 March 1997.
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