Glucagon response to exercise is critical for
accelerated hepatic glutamine metabolism and nitrogen
disposal
Mahesh G.
Krishna,
Robert H.
Coker,
D. Brooks
Lacy,
Bradley
A.
Zinker,
Amy E.
Halseth, and
David H.
Wasserman
Department of Molecular Physiology and Biophysics and Diabetes
Research and Training Center, Vanderbilt University School of Medicine,
Nashville, Tennessee 37232
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ABSTRACT |
The aim of this
study was to determine the role of glucagon in hepatic glutamine (Gln)
metabolism during exercise. Sampling (artery, portal vein, and hepatic
vein) and infusion (vena cava) catheters and flow probes (portal vein,
hepatic artery) were implanted in anesthetized dogs. At least 16 days
after surgery, an experiment, consisting of a 120-min equilibration
period, a 30-min basal sampling period, and a 150-min exercise period,
was performed in these animals. [5-15N]Gln was infused
throughout experiments to measure gut and liver Gln kinetics and the
incorporation of Gln amide nitrogen into urea. Somatostatin was infused
throughout the study. Glucagon was infused at a basal rate until the
beginning of exercise, when the rate was either 1) gradually
increased to simulate the glucagon response to exercise
(n = 5) or 2) unchanged to maintain basal glucagon (n = 5). Insulin was infused during the
equilibration and basal periods at rates designed to achieve stable
euglycemia. The insulin infusion was reduced in both protocols to
simulate the exercise-induced insulin decrement. These studies show
that the exercise-induced increase in glucagon is 1)
essential for the increase in hepatic Gln uptake and fractional
extraction, 2) required for the full increment in
ureagenesis, 3) required for the specific transfer of the
Gln amide nitrogen to urea, and 4) unrelated to the increase
in gut fractional Gln extraction. These data show, by use of the
physiological perturbation of exercise, that glucagon is a
physiological regulator of hepatic Gln metabolism in vivo.
nitrogen; dog; exertion; liver; amino acid
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INTRODUCTION |
GLUTAMINE (Gln), the
most abundant amino acid in the body, is not only a substrate for
gluconeogenesis but also a major vehicle for nitrogen delivery. The
importance of hepatic Gln metabolism is exemplified by studies in the
exercising dog, during which hepatic Gln uptake increases by twofold
because of an increase in hepatic fractional extraction
(8). Moreover, exercise causes an ~80% increase in the
conversion of Gln to urea, because essentially all of the amide
nitrogen on the Gln taken up by the liver goes to the formation of urea
(8).
Despite the important role of this amino acid in nitrogen metabolism,
very little is known about how hepatic Gln metabolism is regulated in
vivo. The hypothesis tested in the present study is that the increase
in glucagon is the signal that determines the exercise-induced increase
in hepatic Gln uptake and incorporation of the amide nitrogen to urea.
This is based on the demonstration that glucagon acutely stimulates net
Gln uptake and flux through the glutaminase step in the isolated,
perfused rat liver (13), increases glutaminase activity in
hepatocytes (3), and rapidly stimulates the formation of
[15N]urea from [15N]Gln (21).
A role for the rise in glucagon in the exercise-induced increase in the
net hepatic fractional extraction of alanine has already been shown
(26).
In the present study, the role of the increase in circulating glucagon
during exercise was tested in the chronically catheterized dog.
Somatostatin was infused intravenously to suppress endogenous pancreatic hormone release, and insulin and glucagon were replaced at
the rates necessary to mimic or prevent the increase in glucagon secretion normally seen during exercise. Hepatic nitrogen metabolism was quantified using stable isotopic and arteriovenous difference methods.
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MATERIALS AND METHODS |
Animal maintenance and surgical procedures.
Mongrel dogs (n = 10; mean wt 24.5 ± 0.8 kg) of either gender that had been fed a standard diet (Kal Kan beef
dinner, Vernon, CA and Wayne Lab Blox: 51% carbohydrate, 31% protein,
11% fat, and 7% fiber based on dry weight, Allied Mills, Chicago, IL)
were studied. Dogs were fed once daily. The total energy intake of this
diet corresponded to 85 kcal · kg
1 · day
1, and the
absolute protein intake was 6 kcal · kg
1 · day
1. In dogs
maintained on this diet, body weight is unchanged from the day of
surgery to the day of the experiment. The dogs were housed in a
facility that met American Association for Accreditation of Laboratory
Animal Care guidelines, and the protocols were approved by Vanderbilt
University's Institutional Animal Care and Use Subcommittee.
At least 16 days before each experiment, a laparotomy was performed
under general anesthesia (0.04 mg/kg of atropine and 15 mg/kg pentothal
sodium presurgery and 1.0% isoflurane inhalation anesthetic during
surgery). Silastic catheters (0.03 in ID) were inserted into the vena
cava and splenic and mesenteric veins for infusions. The tips of the
splenic and mesenteric vein infusion catheters were advanced just
beyond where they coalesce with the portal vein. Silastic catheters
(0.04 in ID) were inserted into the portal vein and left common hepatic
vein for blood sampling. Incisions were also made in the neck region
for the placement of a sampling catheter (0.04 in ID) in the carotid
artery. The carotid artery catheter was advanced so that its tip rested
in the aortic arch. After insertion, the catheters were filled with saline containing heparin (200 U/ml), and their free ends were knotted.
Doppler flow probes (Instrumentation Development Laboratory, Baylor
University School of Medicine) were used to measure portal vein and
hepatic artery blood flows, as described previously (8, 12,
14). The Doppler probe leads and the knotted free catheter ends,
with the exception of the knotted end of the carotid artery catheter,
were stored in a subcutaneous pocket in the abdominal region so that
complete closure of the skin incision was possible. The free end of the
carotid artery catheter was stored under the skin of the neck.
Starting 1 wk after surgery, dogs were exercised on a motorized
treadmill so that they would be familiar with the procedures. Animals
were not exercised during the 48 h preceding an experiment. Only
animals that had 1) a leukocyte count
>18,000/mm3, 2) a hematocrit >36%,
3) normal stools, and 4) a good appetite (consuming all of the daily ration) were used.
On the day of the experiment, with the use of local anesthesia (2%
lidocaine), the subcutaneous ends of the catheters were freed through
small skin incisions made on the subcutaneous pockets in which
catheters were stored. The contents of each catheter were aspirated,
and catheters were flushed with saline. Silastic tubing was connected
to the exposed catheters and brought to the back of the dog, where the
tubing was secured with quick-drying glue. Saline was infused in the
arterial catheter throughout the experiments (0.1 ml/min).
Experimental procedures.
Experiments were performed in 18-h-fasted dogs. All experiments
consisted of a 120-min equilibration period (
150 to
30 min), a
30-min basal sampling period (
30 to 0 min), and a 150-min
moderate-intensity exercise period (0 to 150 min). Exercise was
performed at 100 m/min, 12% grade on a motorized treadmill. The
exercise intensity used in these experiments has previously been shown
to result in a twofold increase in heart rate and an increase in
O2 uptake to 50% of maximum (20). After a
baseline arterial blood sample was obtained, a primed constant-rate
venous infusion of [5-15N]Gln (36 µmol/kg prime, 0.4 µmol · kg
1 · min
1
infusion) was initiated at t =
150 min.
[5-15N]Gln was obtained from Cambridge Isotope
Laboratories (Andover, MA).
Somatostatin was infused into the vena cava beginning at t =
150 min and was continued until the end of the study (0.8 µg · kg
1 · min
1).
Glucagon and insulin were infused into the portal vein also, beginning
at t =
150 min. Glucagon was infused at a basal rate (0.65 ng · kg
1 · min
1) until the
beginning of exercise (0 min), at which time the rate was either
1) gradually increased to simulate the glucagon response to
this exercise protocol (25) (SimG; n = 5)
or 2) unchanged to maintain glucagon levels at basal (BasG;
n = 5). Insulin was infused intraportally at 250 µU · kg
1 · min
1 beginning
at t =
150 min. The insulin infusion rate was then adjusted during the equilibration period, on the basis of feedback from
glucose values obtained by rapid arterial sampling, to achieve stable
euglycemia. The insulin infusion rate at which this stable glucose
level was obtained was 230 ± 33 µU · kg
1 · min
1 in SimG
and 230 ± 25 µU · kg
1 · min
1 in BasG.
As described previously (25), the insulin infusion rate
was adjusted during exercise to simulate the normal exercise-induced fall in insulin in both groups. Arterial glucose was clamped during the
exercise periods using a glucose infusion the rate of which was based
on feedback from small arterial samples. Arterial, portal vein, and
hepatic vein samples were drawn at t =
30,
15, 0, 25, 37.5, 50, 75, 87.5, 100, 125, 137.5, and 150 min. Portal vein and
hepatic artery blood flows were recorded continuously on-line (12).
Processing of blood and tissue samples.
Blood samples were collected in heparinized syringes and put in tubes
containing 1) EDTA for analysis of plasma, 2) 4%
perchloric acid for analysis of whole blood, or 3) EGTA and
glutathione for analysis of plasma catecholamines. Samples were then
centrifuged, and the supernatant was removed. Gln (18),
glutamate (Glu) (18), and urea (17)
concentrations were measured in the supernatant from perchloric
acid-deproteinized blood. Plasma glucose concentration was measured
during the experiments with the glucose oxidase method. Plasma Gln
enrichment, expressed as the molar percent excess, was determined using
the tertiary butyldimethylsilyl derivatives. Derivatized samples were
analyzed on a Hewlett-Packard 5957 gas chromatography-mass spectrometry
system by use of electron ionization and selected ion monitoring of
ions with mass-to-charge ratios of 431/432 for Gln. Plasma urea
enrichment was measured by Metabolic Solutions (Merrimack, NH). For
this purpose, samples were reacted with urease in a sealed tube in
which a trapping well containing H2SO4 was
placed. The NH4 that was formed was released from solution with an alkalinizing agent (5 M NaOH) and trapped in the wells, forming
ammonium sulfate. The N enrichment of an aliquot of the ammonium
sulfate solution was analyzed using isotope ratio mass spectrometry.
Plasma insulin, glucagon, cortisol, and catecholamine analyses were
done by established techniques that have been described previously
(10).
Calculations.
Gln appearance (Ra) in and disappearance (Rd)
from arterial plasma were measured using an isotope dilution method
described by Eqs. 1 and 2, respectively. The
validity of this method has been recently assessed for the Gln system
(16).
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(1)
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(2)
|
R* is the infusion rate of [5-15N]Gln,
Ea is the arterial enrichment of Gln, [Gln] refers to the
blood Gln concentrations, the subscript a is used to designate arterial
blood, V is the volume of Gln distribution, and p is the pool fraction.
V and p are assumed to be 360 ml/kg and 0.75, respectively, on the
basis of work by Kreider et al. (16).
Extrahepatic splanchnic balance is calculated as the difference between
arterial and portal venous blood multiplied by portal venous flow. For
brevity, and because most of this tissue bed is gut, we have defined
extrahepatic splanchnic balances as gut balances. Net gut and liver Gln
balance (NGGlnB and NHGlnB, respectively) were calculated as follows
|
(3)
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(4)
|
where the subcripts pv, ha, and hv refer to portal vein, hepatic
artery, and hepatic vein, respectively, and BF refers to blood flow.
Net Glu balances were calculated in a similar manner. Net hepatic urea
output was calculated by an equation analogous to Eq. 4,
except that the + and
signs were reversed so that positive
numbers would reflect net output. Unidirectional gut (GGlnFE) and
hepatic (HGlnFE) fractional Gln extraction was calculated using
isotopic Gln, such that
|
(5)
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(6)
|
Gln delivery to gut and liver (GGlnD and HGlnD, respectively)
were calculated by the following equations
|
(7)
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(8)
|
Unidirectional gut (GGlnU) and hepatic (HGlnU) Gln uptake from
the blood could then be calculated from the equations
|
(9)
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(10)
|
and unidirectional gut (GGlnO) and hepatic (HGlnO) Gln output
into the blood could be calculated as
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(11)
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(12)
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The rate of conversion of the Gln amide nitrogen into urea was
calculated as
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(13)
|
where V is the volume of urea distribution in the whole body and
is assumed to be 600 ml/kg (19).
Data analysis.
Experiments consisted of four sampling periods. These were the 30-min
basal period and the 25- to 50-min, 75- to 100-min, and 125- to 150-min
exercise periods. Individual samples within a sampling period were
pooled and meaned. The values from each period are presented as the
change from basal. Statistics were performed using SuperAnova (Abacus
Concepts, Berkeley, CA) on a MacIntosh PowerPC. Statistical comparisons
between groups and over time were made using ANOVA designed to account
for repeated measures. Significant differences were assessed using
contrasts solved by univariate repeated measures. Differences were
considered significant when P values were <0.05. Data are
expressed as means ± SE.
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RESULTS |
Arterial glucagon and insulin concentrations.
Plasma glucagon concentrations rose by twofold over the course of the
exercise period in SimG, whereas concentrations were unchanged from
basal in BasG (Fig. 1). Concentrations in
BasG were significantly less than concentrations in SimG from 75 to 150 min (P < 0.01-0.001). Plasma insulin levels fell
similarly in SimG and BasG.

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Fig. 1.
Effect of exercise with somatostatin plus simulated
intraportal insulin and either simulated ( ) or basal
( ) intraportal glucagon on arterial plasma glucagon
(top) and insulin (bottom) concentrations. Data
are means ± SE; n = 5 in each group.
* Significantly different from basal (P < 0.05-0.001); significantly different from corresponding
interval in simulated glucagon protocol (P < 0.01-0.001).
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Arterial glucose concentrations and glucose infusion rates.
Basal arterial glucose concentrations were 109 ± 6 and 109 ± 6 mg/dl in SimG and BasG, respectively. Glucose concentrations were
stable in both groups throughout the exercise period. No exogenous
glucose was required to clamp glucose in SimG, whereas a maximum of
3.1 ± 1.4 mg · kg
1 · min
1 was infused
by the end of exercise in BasG.
Portal vein and hepatic artery blood flows.
Portal vein (basal values of 20 ± 1 and 18 ± 2 ml · kg
1 · min
1 in SimG and
BasG) and hepatic artery blood flows (basal values of 5 ± 1 and
6 ± 1 ml · kg
1 · min
1
in SimG and BasG in the basal state) were unaffected by exercise and
were not significantly different between groups.
Arterial Gln concentrations.
Arterial Gln fell gradually when the glucagon response was simulated
(P < 0.005 at 75-100 min and 125-150 min)
but was unchanged from basal when the glucagon response was maintained
at basal (Fig. 2). Gln concentrations
were significantly higher in BasG than in SimG during the last two
exercise intervals (P < 0.001).

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Fig. 2.
Effect of exercise with somatostatin plus simulated intraportal
insulin and either simulated (hatched bars) or basal (open bars)
intraportal glucagon on arterial blood Gln concentrations. Data are
means ± SE; n = 5 in each group.
* Significantly different from basal (P < 0.005);
significantly different from corresponding interval in simulated
glucagon protocol (P < 0.001).
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Liver glutamine kinetics.
Basal net hepatic Gln balance (Fig. 3)
was
0.5 ± 0.5 and
0.4 ± 0.2 µmol · kg
1 · min
1 in SimG
and BasG, respectively, where a negative value reflects net Gln output.
With the onset of exercise, net hepatic Gln output changed to net
uptake in both groups, a state that remained for the duration of the
exercise period. Net hepatic Gln balance was significantly different
from basal rates during the entire exercise period in both groups
(P < 0.05-0.001). The nature of the change in net
hepatic Gln balance was different in SimG and BasG. In BasG, the peak
rate of net hepatic Gln uptake was obtained within the first sampling
interval. In SimG, the response continued to rise so that the peak
values were reached during the last two sampling intervals. This
created responses that were significantly different between SimG and
BasG at the last two sampling intervals (P < 0.05-0.02).

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Fig. 3.
Effect of exercise with somatostatin plus simulated intraportal
insulin and either simulated (hatched bars) or basal (open bars)
intraportal glucagon on net hepatic Gln balance (top),
unidirectional hepatic Gln uptake (middle), and
unidirectional hepatic Gln output (bottom). The latter two
variables were calculated using hepatic extraction of stable and
naturally occurring isotopes. Data are means ± SE;
n = 5 in each group. * Significantly different from
basal (P < 0.05-0.001); significantly
different from corresponding interval in simulated glucagon protocol
(P < 0.05-0.02).
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Unidirectional hepatic Gln uptake (Fig. 3) rose by sixfold over basal
in SimG by the last exercise interval. Significant increases were
observed at 75-100 min and 125-150 min (P < 0.02-0.001). No significant changes from basal hepatic Gln uptake
were present in BasG. Responses differed between SimG and BasG
during 75-100 min and 125-150 min (P < 0.05-0.02). Unidirectional hepatic Gln output (Fig. 3) was
unaffected by exercise in SimG until the last exercise sampling
interval, during which it was increased compared with basal
(P < 0.05). Hepatic Gln output was unaffected by
exercise in BasG. Hepatic Gln output responses to exercise were not
significantly different between groups (P = 0.062 at
125-150 min).
The exercise-induced increment in glucagon resulted in a gradual
increase in hepatic fractional Gln extraction (Fig.
4) that reached sevenfold basal by the
end of the exercise period. The increment in hepatic fractional Gln
extraction was significantly elevated throughout the exercise period in
SimG (P < 0.05-0.001). In contrast, this variable
was unaffected by exercise when the rise in glucagon was absent.
Responses to exercise were significantly different between SimG and
BasG at the 75- to 100-min and 125- to 150-min intervals.

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Fig. 4.
Effect of exercise with somatostatin plus simulated intraportal
insulin and either simulated (hatched bars) or basal (open bars)
intraportal glucagon on unidirectional hepatic fractional Gln
extraction measured isotopically. Data are means ± SE;
n = 5 in each group. * Significantly different from
basal (P < 0.05-0.001); significantly
different from corresponding interval in simulated glucagon protocol
(P < 0.05).
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Gut Gln kinetics.
Exercise increased net gut Gln balance (Table
1) so that an increase in net uptake was
present in both SimG (P < 0.05-0.01 throughout
the exercise period) and BasG (P < 0.05 at 25- to
50-min and 125- to 150-min intervals). In light of this, it was
surprising that there were no significant changes in either
unidirectional gut Gln output or uptake in either group (Table 1). Gut
fractional Gln extraction was significantly increased with exercise,
regardless of the glucagon replacement protocol (Table 1). Significant
increases were present in both groups at the last two sampling
intervals (P < 0.05-0.01). There were no
significant differences between protocols in any variable related to
gut Gln kinetics.
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Table 1.
Effect of exercise on gut Gln net balance, output, uptake, and
fractional extraction in dogs receiving somatostatin plus
exercise-simulated insulin and glucagon and in dogs receiving
somatostatin plus exercise-simulated insulin and basal glucagon
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Arterial Glu concentrations and net hepatic and gut Glu balances.
Arterial blood Glu concentration was unchanged from basal (66 ± 4 µM) in SimG until the last exercise sampling interval (77 ± 8 µM), at which time it was significantly elevated (P < 0.05). Arterial Glu concentration increased throughout the exercise
period in BasG (P < 0.05-0.001) from a basal
value of 79 ± 8 to 100 ± 8 µM by the end of exercise. The
gut was a net producer of Glu at rest (
0.28 ± 0.04 and
0.24 ± 0.06 µmol · kg
1 · min
1 in SimG
and BasG), and rates of net gut Glu balance were not significantly
affected by exercise or the glucagon replacement protocol. Net hepatic
Glu balance indicated a low rate of net uptake at rest in both groups,
which was not significantly different from zero. Rates were not
significantly changed with exercise in SimG but became net output at
exercise sampling intervals of 25-50 min (
0.08 ± 0.04 µmol · kg
1 · min
1) and
75-100 min (
0.16 ± 0.06 µmol · kg
1 · min
1) in
BasG (P < 0.05). BasG was significantly different from
SimG at the 75- to 100-min sampling interval (P < 0.05).
Arterial urea concentrations and net hepatic urea output.
Arterial urea concentration (Fig. 5) was
unaffected by exercise in SimG until the last sampling interval, at
which time urea was increased (P < 0.001). Arterial
urea concentration was constant throughout the exercise period in BasG.
There were no differences in arterial urea concentration between
groups. Net hepatic urea output (Fig. 6)
rose in response to exercise in SimG by ~10
µmol · kg
1 · min
1
(P < 0.01-0.05 at all exercise sampling
intervals) but was unchanged by exercise in BasG. A significant
difference between groups was present at the 25- to 50-min interval
(P < 0.05). The formation of urea from the Gln amide
nitrogen occurred at a fourfold greater rate in SimG than in BasG (Fig.
7).

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Fig. 5.
Effect of exercise with somatostatin plus simulated intraportal
insulin and either simulated (hatched bars) or basal (open bars)
intraportal glucagon on arterial blood urea concentration. Data are
means ± SE; n = 5 in each group.
* Significantly different from basal (P < 0.001).
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Fig. 6.
Effect of exercise with somatostatin plus simulated intraportal
insulin and either simulated (hatched bars) or basal (open bars)
intraportal glucagon on net hepatic urea output. Data are means ± SE; n = 5 in each group. * Significantly different
from basal (P < 0.05-0.01); significantly
different from corresponding interval in simulated glucagon protocol
(P < 0.05).
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Fig. 7.
Effect of exercise with somatostatin plus simulated
intraportal insulin and either simulated (hatched bar) or basal (open
bar) intraportal glucagon on formation of urea from Gln amide nitrogen
over a 150-min exercise period. Data are means ± SE;
n = 5 in each group. Significantly different from
corresponding rate in simulated glucagon protocol (P < 0.05).
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DISCUSSION |
Arteriovenous and stable isotopic techniques were used to gain an
understanding of the role of glucagon in hepatic glutamine metabolism
during exercise. Exercise is a unique model for studying glucagon
action, because it allows for an increment in glucagon to be studied
without creating hyperglycemia or hyperinsulinemia, both of which may
influence Gln metabolism (1). The absence of these two
factors that suppress hepatic glucagon action allows the full effect of
an increment in glucagon to be assessed. Moreover, the dog model
permits not only the measurement of hepatic balance but also the
replacement of glucagon into its physiological entry site in the portal
vein. This enables glucagon replacement to normal portal vein levels
without creating peripheral hyperglucagonemia. Experiments presented
with the use of these tools clearly illustrate that a physiological
increase in glucagon plays a major role in regulation of hepatic Gln
uptake and incorporation of its amide nitrogen to urea. Gln is one of
two amino acids that are of primary importance for nitrogen delivery
from peripheral tissue to liver. We have shown in previous studies that
the net hepatic uptake and fractional extraction of the other amino
acid, alanine, is predominantly controlled by glucagon during exercise
as well (26). The fact that an increase in glucagon
stimulates both hepatic alanine and Gln uptake highlights the effects
of glucagon stimulation on both system A and system N transporters,
respectively (15), and pathways of metabolism
(27). Unidirectional hepatic Gln uptake and fractional
extraction rose by ~sixfold and ~sevenfold, respectively, during
the last 25 min of exercise when the glucagon response to exercise was
simulated. These variables were unchanged when glucagon concentrations
were maintained at basal throughout the exercise period. The rise in
glucagon during exercise also resulted in a small increase in hepatic
Gln output at the last exercise time interval. This is consistent with
the demonstration that a rise in glucagon stimulates flux through the
Gln synthetase reaction in the perfused rat liver at Gln delivery rates
similar to those of the present study (13).
The finding that the increase in glucagon controls hepatic Gln uptake
is consistent with previous experiments conducted in vitro (3,
13, 21). It has been shown that glucagon leads to an acute
stimulation of net Gln uptake by the isolated perfused rat liver, which
is related to an increase in phosphate-dependent glutaminase activity
(3) and flux through the glutaminase step (13). It was recently shown, moreover, that
[15N]urea formation from [2-15N]- and
[5-15N]Gln is stimulated by glucagon in the perfused rat
liver (3). One result of glucagon-stimulated hepatic Gln
uptake and fractional uptake was that circulating Gln fell during
exercise, whereas it was constant when glucagon levels were not
increased. This is consistent with studies showing that an increment in
glucagon decreases arterial Gln levels in the resting state (2,
6, 23) and increases hepatic gluconeogenesis from Gln
(23).
By comparing net hepatic urea output (Fig. 6) and rate of urea
formation from Gln amide nitrogen (Fig. 7), we can estimate the
importance of Gln amide nitrogen to hepatic ureagenesis. In the group
in which the glucagon response was simulated, ~25% of the urea
nitrogen came from the Gln amide nitrogen. In the basal glucagon group,
the contribution was somewhat lower at ~10 to 15%. When we consider
the number of amino acids that could potentially play a role in
nitrogen delivery, that any one amino acid contributes as much as 25%
under physiological conditions clearly makes it an important vehicle in
nitrogen transport and metabolism.
It is well known that Gln is a major oxidative fuel for the small
intestine (29). This is reflected by the presence of net and unidirectional gut Gln uptake in basal and exercise states. These
data also confirm earlier studies showing that exercise increases net
(24) and unidirectional (8) gut fractional Gln extraction. In the present study, gut fractional Gln extraction rose by two- to threefold. The mechanism for this increase does not
involve the increment in glucagon, because it occurs even when the
glucagon response to exercise is abolished. From a functional standpoint, it is unclear as to whether this increase in gut fractional Gln extraction is to provide more energy for the gut during exercise or
whether it is to facilitate the removal of circulating nitrogenous compounds. This increase in gut fractional extraction decreases the
need to have the liver take up Gln. The increase in net gut fractional
Gln extraction has been shown to correspond to an increase in net gut
ammonia output (24). Because increased portal vein ammonia
stimulates Gln uptake in the liver, it is also possible that increased
gut ammonia released from intestinal Gln metabolism could facilitate
Gln uptake in the liver (4).
Despite alterations in gut and liver Gln kinetics during exercise,
arterial blood Gln kinetics are changed very little or not at all
(8). Moreover, even though the increase in glucagon caused
marked differences in hepatic Gln kinetics in the present study, there
was no effect on arterial Gln turnover. These findings suggest that
arterial blood samples are relatively insensitive to acute changes in
splanchnic Gln metabolism. The reason for this may relate to an
inability of a vena cava isotope infusion to accurately mimic
splanchnic Gln production and utilization. This is consistent with
studies conducted in human subjects, where changes in Gln turnover have
not been detectable at times when changes might be anticipated. In two
separate studies, an infusion of glucagon that caused decrements in
arterialized blood Gln levels did not significantly change measured
rates of Gln appearance or disappearance (2, 23). In one
of these studies, changes in Gln appearance and disappearance were not
present, even though glucagon stimulated gluconeogenesis from Gln
(23). In another instance, no difference in arterial Gln
turnover was evident in exercising human subjects (28),
even though increased regional Gln kinetics have been reported to occur
(7, 8). Nevertheless, the modified one-compartment isotope
dilution method has been shown to accurately trace increases in Gln
infusion rate by use of Gln labeled with isotopes of nitrogen or carbon
(16). One cannot rule out the possibility, therefore, that
reciprocal changes occurring in other tissues (e.g., kidney and muscle)
are enough to counterbalance the kinetic changes of Gln in the
splanchnic bed. These data also emphasize the difficulty of
interpreting isotopic Gln data by use of systemic sampling alone.
Exercise leads to an increase in net hepatic urea output in the dog
(8, 24). In contrast, stable isotope-determined urea production was unchanged in human subjects during 3 h of light exercise (40% maximum O2 uptake) and 1 h of heavy
exercise (70% maximum O2 uptake) (5). This
discrepancy may occur because urea isotope dilution methods have been
been shown to dramatically underestimate acute changes in urea
production assessed using [13C]urea and calculated using
a one-compartment model (9). It may be that the urea space
is sufficiently large that it is difficult to trace in the whole body.
Studies using other indexes of urea production (i.e., changes in urea
excretion, blood levels, and sweat loss) have been shown to increase
with exercise (11, 22). The results of the present study
show that the exercise-induced increase in net hepatic urea output is
reliant on the increase in glucagon levels. Moreover, prevention of the
increase in glucagon resulted in a rate of hepatic conversion of Gln to
urea during exercise that was only 25% of the normal response.
Glucagon has been shown to stimulate ureagenesis in perfused rat liver
(13). Furthermore, this hormone was demonstrated to
increase the incorporation of 15N-labeled amide nitrogen
into urea in isolated rat hepatocytes (3) and perfused rat
liver (21). The mechanism for the glucagon-stimulated increase in urea formation appears to be related to an increase in
glutaminase activity and an accelerated formation of
N-acetylglutamate, an activator of the key ureagenic enzyme,
carbamoyl-phosphate synthetase (3).
Finally, it is important to note the difficulty in measuring hepatic
balances of cold and isotopic substrates. The signal in balance
measurements is the arteriovenous difference, which is generally only a
fraction of absolute concentrations. The measurements of Gln and urea
balances are particularly vulnerable, because hepatic arteriovenous
differences are small, whereas circulating concentrations are high.
This creates a particularly low signal-to-noise ratio. We have worked
to minimize this problem by pooling samples within an exercise
interval. Regardless, one must be particularly wary, under these
conditions, of the possibility of missing significant differences when
they may truly exist (type I statistical error).
In conclusion, these data confirm that Gln is an important vehicle for
nitrogen transport to the liver and show that the exercise-induced increase in glucagon is critical for hepatic Gln metabolism. These data
specifically show that the exercise-induced increase in glucagon is
1) essential for the increase in hepatic Gln uptake and
fractional extraction, 2) required for the full increment in
ureagenesis, 3) required for the specific transfer of the
Gln amide nitrogen to urea, and 4) unrelated to the increase
in gut fractional Gln extraction. These data show, for the first time
by use of the physiological perturbation of exercise, that glucagon is
a physiological regulator of hepatic Gln metabolism in vivo.
 |
ACKNOWLEDGEMENTS |
We acknowledge the valuable assistance of Dr. Paul Flakoll and Li
Zheng for measurements of glutamine enrichment; Eric Allen, Pam Venson,
and Wanda Snead for hormone measurements; and Robert Allison for
technical assistance during experiments.
 |
FOOTNOTES |
This research was supported by National Institute of Diabetes and
Digestive Diseases Grants R01-DK-47344, R01-DK-50277, and Diabetes
Research and Training Center Grant 5-P60-DK-20593.
Address for reprint requests and other correspondence: D. H. Wasserman, Light Hall Rm. #702, Dept. of Molecular Physiology and
Biophysics, Vanderbilt Univ. School of Medicine, Nashville, TN
37232-0615 (E-mail:
david.wasserman{at}mcmail.vanderbilt.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 21 July 1999; accepted in final form 25 April 2000.
 |
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