Interaction David H. Wasserman,
of Gut and Liver in Nitrogen Richard J. Geer, Phillip E. Williams,
Metabolism
During
Exercise
Teresa Becker, D. Brooks Lacy, and Naji N. Abumrad
The role of the gut and liver in nitrogen metabolism was studied during rest, 150 minutes of moderate-intensity treadmill exercise, and 90 minutes of recovery in 18 hour-fasted dogs (n = 6). Dogs underwent surgery 16 days before an experiment for implantation of catheters in a carotid artery and in the portal and hepatic veins, and Doppler flow cuffs on the hepatic artery and portal vein. Arterial glutamine, alanine, and q-amino nitrogen (AAN) levels decreased gradually with exercise (P < .05), while arterial glutamate, NH,, and urea were unchanged. Net gut glutamine uptake was 1.3 2 0.5 pmol/kg . min at rest, and increased transiently to 2.5 2 0.3 +mol/kg min at 60 minutes of exercise (P < .05) as gut extraction increased. Net hepatic glutamine uptake was 0.6 2 0.4 kmol/kg min at rest, and increased to 3.4 f 0.6 and 2.6 + 0.5 kmol/kg . min after 60 and 150 minutes of exercise (P < .05) as hepatic extraction increased. Net gut glutamate and NH, output both increased transiently with exercise (P .: .05). These increases were matched by parallel increments in the net hepatic uptakes of these compounds. Alanine output by the gut and uptake by the liver were unchanged with exercise. Net gut AAN output was -2.1 ? 1.8 pmol/kg min at rest (uptake occurred), and increased transiently to 11.2 * 3.5 pmol/kg min after 30 minutes of exercise (P < .05). Net hepatic AAN uptake was 6.9 2 1.9 Fmol/kg . min at rest, and increased to 21.7 2 4.0 and 18.5 2 2.7 kmol/kg min after 30 and 150 minutes of exercise (P i .02). Net hepatic urea output increased from 3.8 2 0.8 pmol/kg min at rest to 6.8 ? 1.0 and 5.6 ? 1.0 pmol/kg min after 30 and 150 minutes of exercise (P < .05). In summary, during exercise, (1) glutamine uptake by the gut and liver is increased as fractional extraction rises; (2) gut amino acid and ammonia output occur in synchrony with, but exceed gut glutamine uptake; (3) hepatic glutamate and NH, uptake increase in parallel with the increased gut output of these substrates and appear to be closely regulated by their hepatic delivery rates; and (4) hepatic urea output is increased with the increase in hepatic uptake of nitrogenous precursors, but is quantitatively insufficient to explain the entire fate of these nitrogenous compounds within the liver. These studies demonstrate that gut and liver interactions play an important role in nitrogen metabolism during exercise. Copyright (il 199 1 by W. 6. Saunders Company
A
CCELERATED RATES of branched chain amino acid oxidation’.’ and adenosine monophosphate deaminatrot? lead to an increased production of ammonia in working muscle. The synthesis of glutamine’, and alanine’ appear to provide the chief vehicles by which excess ammonia can be shuttled away from this site. However, little is known about the subsequent metabolism of these amino acids during exercise, or the fate of the nitrogen that is liberated during the extramuscular breakdown of these amino acids. IJnder basal conditions, the splanchnic bed provides the primary site of glutamine and alanine removal.’ In the immediate postabsorptive state, glutamine is consumed by the gut and the carbon skeleton is oxidized or released into the portal vein as glutamate. The ammonia released in these reactions appears in the portal vein as such or is used in the synthesis of other amino acids. The liver is generally a net consumer of both alanine and glutamine,8,9 but under certain conditions glutamine may be released from this organ (eg, starvation,“’ acidosis”). When these amino acids are extracted by the liver, their carbon skeletons are either oxidized or used as precursors in gluconeogenesis and the nitrogen is utilized in ureagenesis or in the synthesis of amino acids and nucleotides.’ During exercise, the increased rate of muscle ammonia formation creates a greater importance for the avenues of glutamine and alanine disposal and nitrogen metabolism. The purpose of this study was to examine the magnitude and mechanism by which the gut and the liver interact in the metabolism of nitrogenous compounds during exercise. MATERIALS
Animals
and Sqicaf
AND METHODS
Procedures
Studies were performed on six mongrel dogs (mean weight, 21.4 t 1.0 kg) of either sex. that were maintained on a standard Merabolism,
Vol40,
No 3 (March).
1991:
pp 307-314
diet (Kal Kan beef dinner, Vernon, CA; and Wayne Lab Blox, St Louis, MO: 51% carbohydrate, 31% protein, 11% fat, and 796 fiber based on dry weight). Sixteen days before the experiment, a laparotomy was performed under general anesthesia (sodium pentobarbital, 25 mg/kg) and silastic sampling catheters (0.04 id) were inserted into the portal vein, the left common hepatic vein. and in a carotid artery. After their insertion, the catheters were filled with saline containing heparin (200 U/mL; Abbott Laboratories, North Chicago, IL) and their free ends were knotted. Doppler flow probes (obtained from Instrumentation Development Laboratory, Baylor University School of Medicine) were used to measure portal vein and hepatic artery blood flow.” Briefly. a small section of the portal vein, upstream from its junction with the gastroduodenal vein, was cleared of tissue, and the appropriate size flow cuff was placed around the vessel and secured. The gastroduodenal vein was isolated and then ligated proximal to its coalescence with the portal vein. Next, a section of the main hepatic artery lying proximal to the portal vein was isolated and the appropriate-size flow cuff was placed around the vessel and secured. The Doppler flow cuff leads and the knotted portal and hepatic vein catheter ends were placed in a subcutaneous pocket near where they exit from the abdominal cavity, and the knotted end of the carotid catheter was placed in a subcutaneous pocket in the ventral neck
From the Departments of Molecular Phwiolop and Blop!zysics. Surgery. and the Diabetes Research and Training Center, khnderbilt University School of Medicine, Nashville, TN. Supported by National Institutes of Health Grunt No. ROl DK42488, Diabetes Research and Training Grant No. SP60-AM-20593-08, and Clinical Nutrition Suppoti Unit Grant No. DK26657. D.H. W. ib a recipient of a Career Development Award fLom the Jwwwile Dlnbetrs Foundation International. Address reprint requests to David H. Wussemtan, PhD. Depaltmertt of Molecular Physiology and Bioph_vsks, Vanderbih (Jnitjersig School of Medicine, Nashville. TN 37232. Copyright c8 1991 by W.B. Saunders Compum 0026049519114003-0016$03.00l0 307
308
region. This procedure permitted complete closure of the skin incisions. Starting 1 week after surgery, dogs were conditioned to run on a motorized treadmill. The exercise duration and intensity were both increased progressively until a dog could perform the exercise parameters used in an experiment. Dogs were not exercised during the 48 hours preceding an experiment. Three days before each experiment, blood was withdrawn to determine the leukocyte count and the hematocrit of the animal. Only animals that had (1) a leukocyte count below 18,OOO/~L,(2) a hematocrit greater than 38%, (3) a good appetite (consuming all of the daily ration), and (4) normal stools, were used. On the day of the experiment, after an 18-hour fast, the free ends of the catheters were freed through a small skin incision made under local anesthesia (2% lidocaine, Astra Pharmaceutical, Worcester, MA). The contents of each catheter were aspirated, and the catheters were flushed with saline. Silastic tubing was connected to the exposed catheters and they were then brought over the back of the dog where they were secured with quick-drying glue allowing for convenient sampling. Saline was infused through the arterial catheter at a slow rate (0.1 mUmin) throughout the experiment. Experimental Procedures Experiments consisted of a control period (-40 to 0 minutes), a period of moderate-intensity (100 m/min, 12% grade) exercise (0 to 150 minutes), and an exercise recovery period (150 to 240 minutes. The work intensity utilized resulted in an increase in heart rate from 91 2 5 to 189 + 9 bpm. Indocyanine green was infused (0.1 mg/m’ min) starting 80 minutes before the control period and was continued for the duration of the experiment. This was used to obtain measurements of total hepatic blood flow to substantiate the determinations obtained with the Doppler flow cuffs, and to verify that the hepatic vein catheter was positioned correctly. Processing of Blood Samples Blood samples were collected from the carotid artery, portal vein, and hepatic vein in heparinized syringes and distributed into tubes containing EDTA (5 mg) or deproteinized in 4% perchloric acid (0.5 mL whole blood in 1.5 mL perchloric acid) and centrifuged. For the measurement of glutamine and glutamate, an aliquot of deproteinized blood from each sample was placed in each of two tubes containing 0.2 mol/L sodium acetate buffer. In one tube glutamine was completely reacted to glutamate with glutaminase (Sigma Pharmaceuticals, St Louis, MO). The amount of glutamate was then determined in both samples by measuring the conversion of NAD to NADH fluorometrically following the reaction catalyzed by glutamate dehydrogenase (Sigma Pharmaceuticals). The glutamine concentration was determined as the difference in glutamate concentrations between the tube that was reacted with glutaminase and the tube that was not. Blood n-amino nitrogen (AAN) levels were measured using o-phthaldehyde and measuring fluorescence in the presence of 2-mercaptoethanol.” Gomparisons in our laboratory show that values obtained using this method for the measurement of AAN levels agree within 5% to values obtained for total amino acids using a modification of the method of Heinrikson and Meridith.“’ Plasma alanine concentration was determined by reaction with ninhydrin. Plasma urea nitrogen was measured calorimetrically at 520 nm by a modification of the urea-diacetyl reaction.” Plasma ammonia levels were determined by the Berthiot reaction.16 Indocyanine green levels were determined spectrophotometrically (805 nm) in arterial and
WASSERMAN
ET AL
hepatic vein plasma samples immediately on completion of the study.
Calculations The net hepatic alanine, glutamine, glutamate, ammonia, and AAN balances were determined by the formula (4. A + P, P)H, H, where A is the arterial concentration, P the portal vein concentration, and H is the hepatic vein concentration. A,, P,, and H, are the arterial, portal vein, and total (A + P,) hepatic blood (plasma) flows as determined by Doppler flow probes. Plasma flow was determined as Blood Flow (1 - hematocrit). Splanchnic balances of these compounds were calculated as Hr. (A - H). The net hepatic balance of urea was calculated as H, H (A, A + P, . P). The hepatic fractional extraction of a compound equals its hepatic uptake divided by its rate of delivery to the liver (A. A + P, P). The net gut balance of a compound was calculated by the formula (A - P) . P,. The fractional extraction of a compound by the gut was calculated by dividing its uptake by the rate of delivery to the gut (P,. A). Glutamine, glutamate, urea, ammonia, and AAN balances were calculated using blood flow measurements and alanine balances were determined based on plasma flow rates. Statistical assessment of the basal versus exercise and recovery periods were performed initially using ANOVA for repeated measures. When the ANOVA yielded significant F values (P < .05), paired t tests were performed.” The statistics presented are the results of the post hoc t tests comparing basal with specific exercise and recovery values. Data are expressed as mean ? SE.
RESULTS
Glutamine Levels, Net Gut, Hepatic, and Splanchnic Balances, and Gut and Hepatic Fractional Extractions Arterial blood glutamine decreased gradually from levels of 665 I~I 31 kmol/L at rest to 557 ? 40 by the end of exercise (P < .05) (Fig 1). Portal and hepatic vein blood glutamine decreased initially with the onset of exercise from levels of 616 + 32 and 635 2 30 pmol/L to levels of 519 2 38 (P < ,005) and 489 + 48 (P < .Ol) pmol/L, respectively, after 60 minutes of muscular work (Fig 1). There were no further decreases in portal or hepatic vein blood glutamine after 60 minutes of exercise (Fig 1). Net gut glutamine uptake increased from a rate at rest of 1.4 2 0.5 to 2.5 ~fi:0.3 kmol/kg . min after 60 minutes of exercise (P < .05), and then declined throughout the remainder of the exercise period (Fig 1). The increase in net gut glutamine uptake was due to an increase in fractional extraction from a value of 0.08 -C 0.03 at rest to 0.16 * 0.02 after 60 minutes of exercise (P < .05) (Table 1). Net hepatic glutamine uptake increased from a rate of 0.6 ? 0.4 kmol/kg . min at rest to a peak of 3.4 ? 0.6 kmol/kg . min after 60 minutes of exercise (P < .05). This increase in uptake was due to an increase in hepatic fractional glutamine extraction from 0.03 * 0.02 at rest to a peak at 60 minutes of exercise of 0.20 + 0.05 (P < .02). Net splanchnic glutamine uptake increased immediately with the onset of exercise from a resting rate of 1.9 2 0.6 pmol/kg min to a peak of 5.9 + 0.7 pmol/kg . min at 60 minutes of exercise (P < .02) (Table 2).
EXERCISE
AND
SPLANCHNIC
1
NITROGEN
METABOLISM
309
A
EXERCISE
I 1
Glutamate Levels and Net Gut, Hepatic, and Splanchnic Balances and Hepatic Fractional Extraction
i
ARTERY
Arterial and hepatic vein blood glutamate levels were 42 t 7 and 44 ? 7 umol/L at rest and were essentially unchanged through exercise and recovery (Fig 2). Portal vein blood glutamate was 40 ? 6 umol/L at rest and increased transiently to a peak level of 56 _t 8 umol/L after 10 minutes of exercise (P < 0.05) (Fig 2). At rest, there was no net gut glutamate exchange (0.07 * 0.05 t.J,mol/kg min). Net gut glutamate output was evident transiently (-0.28 2 0.07 umol/kg . min at 30 minutes of exercise; P < .02). Net hepatic glutamate balance was not significantly different from 0 at rest (-0.09 t 0.08 umol/ kg. min), but transient hepatic glutamate uptake occurred with the onset of exercise (0.31 ? 0.12 kmol/kg min at 30 minutes; P < .05) (Tables 1 and 2).
B
Alanine Levels, Net Gut, Hepatic, and Splanchnic Balances. and Hepatic Fractional Extraction
-60
0
-30
30
60
90
120
150
180
210
Arterial plasma alanine levels were 346 f 20 kmol/L at rest and decreased to 238 2 13 umol/L by the end of exercise (P < .002) (Fig 3). Portal vein plasma alanine levels were 422 t 31 umol/L at rest, increased insignificantly with exercise, initially obtaining a level of 458 2 34 kmol/L at 30 minutes, after which levels decreased gradually to 292 2 18 FmoliL by the end of exercise (P < .Ol). Hepatic vein plasma alanine was 299 ? 20 pmol/L at rest and decreased to 101 +- 11 umol/L by the end of exercise (P < .002). The net gut balance of alanine was - 1.4 2 0.2 ).r,mol/kg . min at rest and -I .O 2 0.2 t.r,mol/kg . min at 150 minutes of exercise. Net hepatic alanine uptake was 2.5 + 0.3 kmolikg . min at rest and was essentially unchanged with exercise (3.1 2 0.4 p,mol/kg min after 30 minutes). Net hepatic fractional alanine extraction increased from 0.25 ? 0.02 at rest to 0.65 & 0.03 by the end of exercise (P < .OOl) (Table 1). Net splanchnic alanine uptake increased gradually from 1.1 2 0.1 to 2.1 I~I0.3 kmol/kg min (P < .05) (Table 2).
240
TIME (min)
AAN Levels, Net Gut, Hepatic, and Splanchnic Balances, Fig 1. (A) Blood arterial, portal vein, and hepatic vein glutamine concentrations and (B) net gut and (12) hepatic glutamine uptake during rest, 150 minutes of treadmill exercise, and 90 minutes of exercise recovery. Data are mean ‘_ SE; n = 6.
and Hepatic Fractional Extraction
Arterial, portal vein, and hcpatic vein AAN levels were 5,448 * 171, 5,375 t 213, and 5,179 t 192 ~mol/L at rest
Table 1. Gut and Hepatic Fractional Extraction
-
Exercise (time in minutes)
Gut fractional Glutamine Hepatic
-
30
10
Basal
Recovery (time in mfnutes)
90
60
150
120
240
180
160
extraction 0.08 2 0.03
fractional
0.13 + 0.02*
0.14 -C 0.03
0.16 t 0.02”
0.12 k 0.02
0.08 k 0.02
0.07 z 0.03
0.08 + 0.01
0.11 2 0.06
0.13 k 0.03
extraction
Glutamine
0.03 + 0.02
0.06 + 0.03
k 0.02*
0.16 t 0.04*
0.13 2 0.04*
0.06 + 0.03
0.10 2 0.02
0.06 2 0.07
0.12 k 0.08
0.16 k 0.03* 0.15 -c 0.06*
0.14
0.0
0.15 k 0.03* 0.20 + 0.07’
0.20 5 0.05’
Glutamate
0.05
2 0.09
Alanine
0.25 _f 0.02
0.29 -c 0.05
0.35 k 0.04*
0.38 k 0.06*
0.50 2 0.05’
0.59
-c 0.05*
0.04 + 0.08 0.65 k 0.03*
0.12 2 0.05 0.61 i 0.04*
0.07 k 0.04 0.60 k 0.04*
0.0 0.64 2 0.04*
Ammonia
0.39 + 0.08
0.47
k 0.10
0.50 2 O.ll*
0.46 2 0.09
+ 0.10
0.38
f 0.10
0.38
2 0.09
0.37
AAN
0.04 t 0.01
-
0.13 2 0.02’
-
-
0.13
-c 0.02*
NOTE.
Data are mean
*P < .05 compared
-c SE; n = 6. No net glutamate
with the basal state.
extraction
0.41
0.13 2 0.03’ was evident
in the basal period
+ 0.10
0.35 + 0.10
0.40 2 0.09
-
0.09 r 0.02*
0.05
or after 90 minutes
of exercise
recovery.
2 0.05
310
WASSERMAN
ET AL
Table 2. Net Splanchnic Balances Exercise Basal
Glutamine Glutamate
10
1.9 + 0.6 -0.02
30
3.6 f 0.8’
r 0.07
-0.01
(time
5.4 + 1.3*
90
5.9 2 0.7*
-
10.6?6.4
are in pmol/kg
Data are mean
min.
-
0.11 -c 0.03
0.10 f_ 0.03
0.11 t_ 0.02
2.11 2 0.27’
2.61 2 0.36’
2.50 + 0.51’
2.61 + 0.41*
Negative
numbers
3.6 f 0.6’ -0.06?
14.7 + 7.7 correspond
3.5 k 0.6’
17.2 + 8.7*
to splanchnic
output,
while
positive
numbers
7.1 + 6.8
10.0 ? 6.1
correspond
to splanchnic
-+ SE; n = 6. with the basal state.
/
*O r
0.12 + 0.03
1.87 + 0.26*
1.75 ? 0.24*
1.45 2 0.41
*P < .05 compared
0.12 + 0.03
0.13 2 0.03
0.16
1.43 ‘- 0.49
uptake.
3.8 + 0.6* 0.01 + 0.16
t 0.05
0.14 2 0.04
Balances
3.1 + 1.1
0.01 + 0.10
1.02 f 0.34
240
0.11 2 0.16
0.32 ? 0.10
0.15 z 0.04
in minutes)
160
0.14 2 0.16
3.2 + 0.5*
+ 0.07
1.07 * 0.12
(time
160
0.25
4.8 k l.l*
0.17
0.12 5 0.03
NOTE.
150
0.03 2 0.17
Alanine
9.0 f 2.7
Recovery 120
+ 0.13
Ammonia AAN
in minutes)
60
and levels in all vessels tended to decrease with exercise (Fig 4). However, levels decreased significantly with exercise only in the artery and hepatic vein (P < .05). Net gut AAN balance was not significantly different from 0 (2.1 2 2.8 kmol/kg . min), but AANs were produced during exercise (-11.2 ‘_ 3.5 kmol/kg . min at 30 minutes of exercise; P < .OS). Net hepatic AAN uptake was evident at rest (6.9 ? 1.9 pmol/kg . min) and increased with exercise (2117 * 4.0 and 18.5 t 2.7 pmol/kg . min after 30 and 150 minutes; P < .02). The increase in hepatic AAN uptake during exercise was due to a threefold increase in fractional extraction (0.04 ? 0.01 at rest and 0.13 * 0.02 after 1.50 minutes of exercise; P < .05) (Table 1). Net splanchnic
* I
VEIN
HEPATIC
A
EXERCISE
B
500
UPTAKE
I
HEPATIC
VEIN
II
L z
-0.4
’ -60
’
’
’
-30
0
30
60
90
TIME
’
’
120
150
180
’
’
210
240
(min) Fig 2. (A) Blood arterial, portal vein, and hepatic vein glutamate concentrations and (B) net gut and (C) hepatic glutamate balance during rest, 150 minutes of treadmill exercise, and 90 minutes of exercise recovery. Data are mean t SE; n = 6.
-61 -60
/ -30
0
30
60
’ 90
J 120
’
150
180
210
240
TIME (min)
Fig 3. (A) Plasma arterial, portal vein, and hepatic vein alanine concentrations and (B) net gut and hepatic alanine balance during rest, 150 minutes of treadmill exercise, and 90 minutes of exercise recovery. Data are mean + SE; n = 6.
EXERCISE AND SPLANCHNIC NITROGEN METABOLISM
311
A
EXERCISE
ammonia extraction was 0.39 + 0.08 at rest and increased with exercise to a peak of 0.50 * 0.11 at 30 minutes of exercise (P < .05) (Tables 1 and 2). Urea Levels and Net Hepatic Output
Plasma urea levels did not change significantly from rest through exercise in the artery, portal vein, and hepatic vein (Table 3). Arterial and portal plasma urea levels were similar from rest through exercise and recovery, indicating that the gut was not a net consumer or producer of this compound. Hepatic vein urea levels were higher in the hepatic vein than in the artery and portal vein throughout the experiment (Table 3). The urea concentration gradient across the liver (hepatic vein concentration minus arterial and portal vein concentrations) increased by twofold from 0.12 2 0.03 t.r,mol/L at rest to 0.23 * U.04 pmol/L at 30 minutes of exercise (P < .05). Net hepatic urea output increased from 3.6 ‘: 0.5 kmol/kg . min at rest to a peak rate of 6.8 t 0.7 kmol/kg min at 30 minutes of exercise (P < .05). Rates decreased gradually for the remainder of the exercise period (Fig 6). Hepatic Artery and Portal Vein Blood Flow I I I
1
I,, -:c
0
30
60
90
120
150
IPJO
210
240
Hepatic artery blood flow was 6.7 & 0.3 and 4.9 ? 0.6 mL/kg . min at rest and by the end of exercise, respectively
TIME (hl) (A) Blood arterial, portal vein, and rkpatic vein AAN concentrations and (B) net gut and hepatic AAN balances during rest, 150 minutes of treadmill exercise, and 90 minutes of exercise recovery. Data are mean rt SE; n = 6.
A
EXERCISE
8or 60 t
AAN uptake was evident at rest and increased gradually with exercise (Table 2). Differences between rest and exercise were significant only at 150 minutes of exercise. Anunonia Levels, Net Gut, Hepatic, and Splanchnic Baiances, and Hepatic Fractional Extraction
Arterial and hepatic vein plasma ammonia levels were 27 2 6 and 23 +- 5 p,mol/L at rest, respectively, and were unaffected by exercise (Fig 5). In contrast, portal vein plasma ammonia levels increased transiently from 40 ? 6 to 54 z 7 pmol/L after 30 minutes of exercise (P < .05). The gut released ammonia at rest (-0.32 + 0.09 kmol/kg min) and this release was accelerated transiently with exercise (-0.65 -c 0.21 Fmol/kg min at 30 minutes of exercise) (Fig 5). Similarly, net hepatic ammonia uptake was 0.46 ? 0.09 kmol/kg . min at rest and increased transiently with exercise (0.79 2 0.21 pmol/kg min at 30 minutes of exercise) (Fig 5). .4lthough gut output and hepatic uptake of ammonia increased with exercise in all dogs, in some animals the peak increments were evident after 10 minutes of exercise, while in others it was evident after 30 minutes. Hence, at no specific time point could significance be detected. However, when the basal period was compared with the transient increment, whether it occurred at 10 or 30 minutes, significance was obtained (P < .05). Net hepatic fractional
PTAk,E __bppI
_,
I -60
z
-30
f
I
0
30
-__
90
60
1:m
_.i
12c
I.>0
_ C’JTPUT
_-.-___-
_i
.50
?:O
240
i ,I !
Fig 5. (A) Plasma arterial, portal vein, and hepatic vein ammonia concentrations and (B) net gut and hepatic ammoniabalance during rest, 150 minutes of treadmill exercise, and 90 minutes of exercise recovery. Data are mean f SE: n = 6.
WASSERMAN
312
Table Exercise Basal
Arterial
10
3.58 2 0.49
3.55
r 0.49
3. Plasma (time
ET AL
Urea Levels Recoven/
in minutes)
30
60
90
3.63 t 0.49
3.65 f 0.48
3.72 2 0.49
120
3.76?
0.51
(time
in minutes)
150
160
180
240
3.58 2 0.54
3.84 -+ 0.53
3.84 t 0.53
3.88 i 0.53
Portal vein
3.60 2 0.49
3.56
r 0.49
3.61 -t 0.48
3.68
2 0.48
3.74 2 0.50
3.80 + 0.52
3.85 + 0.54
3.82 ? 0.54
3.86 + 0.53
3.87 + 0.53
Hepatictiein
3.72 i 0.52
3.75 f 0.51
3.84 2 0.51
3.87
f 0.51
3.94~
4.01
4.05 t 0.56
3.97 f 0.55
3.99 2 0.55
4.00 2 0.57
NOTE.
Levels are in mmol/L
and are expressed
as mean
DISCUSSION
Glutamine and alanine play important roles in the shuttling of nitrogen produced in the working muscle to the splanchnic bed. Glutamine is by far the more important nitrogen carrier, as its splanchnic uptake exceeded that for alanine by 78% at rest and by as much as 300% after 60 minutes of exercise. The importance of glutamine in nitrogen transport is especially apparent since this amino acid contains an amide group in addition to the AAN. The splanchnic uptake of AAN was well in excess of that for the sum of glutamine and alanine uptake, emphasizing the importance of other amino acids in the transfer of nitrogen to the splanchnic bed during rest and exercise. While alanine was consumed solely by the liver, the increased net splanchnic glutamine uptake during exercise was due to a transient twofold increase in net gut glutamine uptake,
EXERCISE
I I
I
z
I I 1
I
I
0 -60-30
I
I I I
I
w
I
0
30
60
90
+ 0.56
+ SE; n = 6.
(P < .05). No significant change in portal vein blood flow was evident with muscular work, as rates were 25.5 f 0.9 and 24.1 ? 2.1 mL/kg . min at rest and by the end of exercise, respectively (Table 4). The rates obtained with the Doppler flow probes yielded values for total hepatic blood flow that were within 10% of those obtained with indocyanine green during rest, exercise, and recovery (data not shown).
I
0.54
120150180210240
TIME (min) Fig 6. Net hepatic urse output during rest, 160 minutes of treadmill exercise, and 90 minutes of exercise recovery. Data are mean f SE; n = 6.
which peaked at 60 minutes of exercise, and to a more sustained increase in net hepatic glutamine uptake. Of the glutamine consumed by the splanchnic bed, approximately 27% was removed by the liver at rest, and approximately 57% and 72% were removed by the liver at 60 and 150 minutes of exercise. Hence, with the transition from rest to exercise, the balance of glutamine uptake by the splanchnic bed shifts from primarily gut to liver utilization. The percentage of the splanchnic glutamine uptake that could be attributed to the liver was reduced toward normal resting values during exercise recovery. The contribution of the liver was 50% of the total splanchnic glutamine uptake 90 minutes after the cessation of exercise. The data from this study indicate that the gut provides the liver with a source of carbon-based substrates for gluconeogenesis (glutamate, alanine, and other amino acids) and nitrogenous compounds (eg, ammonia and AAN) for ureagenesis at an increased rate during exercise. Since the peak increase in gut glutamine uptake coincides with the release of these hepatic substrates, it is likely to serve as a precursor for these compounds. In addition, to the role of gut glutamine metabolism in supplying substrate to the liver, glutamine oxidation may be an important source of energy for the gut.‘8,‘9Interestingly, the AAN output from the gut at 30 minutes of exercise exceeded the net gut uptake of glutamine by threefold. Moreover, the value obtained for net AAN output from the gut underestimates the absolute output due to the uptake of glutamine AAN (and likely other amino acids) by the gut. Hence, it is clear that gut proteolysis or the mobilization of a non-AAN source must be accelerated. Furthermore, these studies introduce the possibility that the gut is a labile substrate store that can be mobilized during exercise. The exercise-induced increase in net gut and hepatic glutamine uptakes occurred despite a decrease in the glutamine delivery to these organs. The entire increment in both net gut and hepatic glutamine uptake was due to an increase in the fractional extraction of this compound. Similarly, hepatic alanine and AAN uptakes were sustained despite decreases in their respective hepatic delivery rates as fractional extraction was increased. The exercise protocol used in this study elicits twofold increases in arterial plasma glucagon, norepinephrine, and epinephrine, and a 50% reduction in insulin.2”~2’Previous studies indicate that the increase in glucagon is essential to the exercise-induced increase in hepatic fractional alanine extraction.‘“” Furthermore, an increase in glucagon, similar to that which occurs with exercise, may lead to an increased extraction of glutamine by the gut and liver?’ In response to exercise, glutamate and particularly ammo-
313
EXERCISE AND SPLANCHNIC NITROGEN METABOLISM
Table 4. Hepatic Arteryand
Portal Vein Blood Flows Recovery (time in minutes)
Exercise (time in minutes) Basal Hepatic
artery
Portal vein
6.7
+
10
0.3
5.3 + 0.6*
25.5 + 1.4 24.3 t 1.5
NOTE. Blood flows are in mUkg lp
< .05 compared
30
5.4 f OS* 24.7 + 2.0
60
5.0 2 0.71 25.0 + 1.8
150
120
5.3 2 0.8
5.0 t 0.6*
25.0 & 2.4 24.2 + 2.3
5.0 t 0.6" 24.1 + 2.1
160
8.8 r 1.4
180
7.5 + 1.5
240
8.5 i-0.9
26.3 + 2.8 26.7 2 2.2 24.8 ? 1.3
min. Data are mean + SE; n = 6.
with the basal state.
were taken up by the liver with nearly the same dynamics as those that characterized the output of these compounds by the gut. Hence, while transient increases in portal vein glutamate and ammonia concentrations were evident within the initial 60 minutes of exercise, no changes in hepatic vein levels of these compounds were evident. Hence, the uptake of glutamate and ammonia were proportional to the rate at which they were delivered to the liver. Net hepatic urea output increased by almost twofold at 30 minutes of exercise to a rate of 6.8 pmol/kg . min. To our knowledge, this is the first direct report of an increase in hepatic urea output during exercise. The peak hepatic ourput of urea in the present study accompanied the peak in hepatic amino acid and ammonia uptakes. This finding is in contrast to work in humans that showed that stable isotope-determined urea production was unchanged over 10.5 minutes of exercise, despite a marked increase in protein breakdown.” Although there is no clear explanation for the discrepancy, it is possible that the relatively light work rate used by these investigators (30% of the maximum oxygen uptake) was insufficient to elicit a detectable increase in urea production. During competitive physical activity, an increase in urea production has been inferred from indirect indices (ie, changes in urea excretion, blood levels, and sweat losses).24.*5An alternative explanation for the apparent absence of an exerciseinduced increase in urea production may pertain to the large urea pool size, which may make isotope dilution methods somewhat insensitive, particularly under nonsteady-state conditions. Nevertheless, validation experiments in the resting, hepatectomized dog have been performed and indicate the measurement of urea turnover with stable isotopes is accurate under steady-state conditions and is generally within ~-20% in the non-steadystate.” Finally, we cannot rule out the possibility that species differences exist in hepatic metabolism, which permit a larger fraction of the nitrogen that is extracted by the liver to be channelled into urea in the dog compared with man. Although hepatic urea output was increased, the uptake of nitrogenous compounds by the liver was still far in excess of rhis rate. We could estimate by summing the hepatic uptakes of AAN, ammonia, and the glutamine amide group, that nitrogen uptake by the liver exceeded the rate at which nitrogen exited the liver via urea by twofold. The fate of the rest of the nitrogen that is extracted by the liver cannot be determined by the results of the present study. However, it has been proposed that hepatic uptake of nitrogenous compounds during exercise may be important for the accelerated formation of acute-phase proteins.”
nia
90
Despite the large increase in hepatic urea output in the present study, only a small gradual increase in arterial urea concentration was evident. This may be due to an increased urea disappearance rate during exercise, or to the large body pool that may effectively dampen the increase in this compound. The calculation of the organ balance of substrates measured in plasma requires assumptions regarding the equilibrium of these substrates between plasma and erythrocytes. Glutamine, glutamate, and AAN were measured in whole blood, and balances were calculated using blood flow: hence no assumptions pertaining to the equilibration across the erythrocyte were necessary. Alanine balances were calculated using plasma concentrations and flows. Similar alanine balances are generally obtained irrespective of whether the calculations are made using plasma or whole blood measurements.L8~Z9The assumption was made in the present study that urea and ammonia rapidly equilibrate across the erythrocyte membrane. Therefore, plasma levels were considered equal to whole blood levels and whole blood flow was used to calculate the gut and hepatic balance of these compounds. In support of this assumption is the report that arterial blood concentrations of both urea and ammonia are within 10% of levels in the plasma and the net balance of these compounds across the perfused dog brain is similar irrespective of whether blood or plasma values are used.‘” In conclusion, during exercise, (1) glutamine plays a greater role in nitrogen transfer from the periphery to the splanchnic bed than in the basal state; (2) gut and liver glutamine uptake is increased independently of load as fractional extraction is increased; (3) the gut AAN output exceeds the gut glutamine uptake, indicating that gut proteolysis may be accelerated; (4) hepatic glutamate and NH, uptakes increase in parallel with the increased gut output of these substrates and appear to be closely regulated by their hepatic delivery rates; (5) net hepatic urea output increases along with the hepatic uptake of nitrogenous precursors; and (6) quantitatively the hepatic uptake of nitrogenous compounds exceeds the output of urea, indicating that hepatic protein synthesis may be accelerated. The results of this study demonstrate the importance of gut and liver interactions in nitrogen metabolism during exercise.
ACKNOWLEDGMENT
The technical assistance of R. Ensley is gratefully acknowledged. The authors illustrations.
are grateful
to D.S. Wasserman
for preparation
of the
WASSERMAN
314
ET AL
REFERENCES
1. Hagg SA, Morse EL, Adibi SA: Effect of exercise on rates of oxidation turnover, and plasma clearance of leucine in human subjects. Am J Physiol242:E407-E410,1982 2. Wolfe RR, Goodenough RD, Wolfe MH, et al: Isotopic analysis of leucine and urea metabolism in exercising humans. J Appl Physiol52:458-466,1982 3. Meyer RA, Terjung RL: AMP deamination and IMP reamination in working skeletal muscle. Am J Physiol239:C32-C38, 1980 4. Eriksson LS, Broberg S, Wahren J: Ammonia metabolism during exercise in man. Clin Physiol5:325-336,1985 5. Felig P, Wahren J, Karl I, et al: Glutamine and glutamate metabolism in normal and diabetic subjects. Diabetes 22:573-576, 1973 6. Felig P, Wahren J: Amino acid metabolism in exercising man. J Clin Invest 50:2703-2714,197l 7. Abumrad NN, Williams P, Frexes-Steed M, et al: Inter-organ metabolism of amino acids in vivo. Diabetes Metab Rev 5:213-226, 1989 8. Miller BM, Cersosimo E, McRae J, et al: Inter-organ relationship of alanine and glutamine during fasting in the conscious dog. J Surg Res 34:310-318,1983 9. Fine A: The effects of chronic metabolic acidosis in liver and muscle glutamine metabolism in the dog in vivo. Biochem J 202:271-273,1982 10. Matsutaka H, Aikawa T, Yamamoto H, et al: Gluconeogenesis and amino acid metabolism. III. Uptake of glutamine and output of alanine and ammonia by non-hepatic splanchnic organs of fasted rats and their metabolic significance. J Biochem 74:10191029,1973 11. Shrock H, Goldstein L: Inter-organ relationship for glutamine metabolism in normal and acidotic rats. Am J Physiol 240:E519-E525,1981 12. Hartley CJ, Hanley HG, Lewis RM, et al: Synchronized pulsed doppler blood flow and ultrasonic dimension measurement in conscious dogs. Ultrasound Med Biol4:99-110,197s 13. Bloomgarden ZT, Liljenquist J, Lacy WW, et al: Amino acid disposition by liver and gastrointestinal tract after protein and glucose ingestion. Am J Physiol241:E90-E99, 1981 14. Heinrikson RL, Meridith SC: Amino acid analysis by reverse phase high pressure liquid chromatography: Pre-column derivatization with phenylisothiocyanate. Anal Biochem 136:65-74,1984 15. Marsh WH, Fingerhut B, Miller H: Automated and manual direct methods for the determination of blood urea. Clin Chem 50:624-627.1965
16. Imler MA, Frick A, Stahl A, et al: Dosage de I’ammoniemie en discontinu et en continupar un technique au dialyse automatique. Clim Chim Acta 37:245-261.1972 17. Zar JH: Biostatistical Analysis. Englewood Cliffs, NJ, Prentice-Hall, 1984 18. Souba WW, Smith RJ, Wilmore DW: Glutamine metabolism by the intestinal tract. JPEN 9:608-617, 1985 19. Windmueller HG, Spath AE: Respiratory fuels and nitrogen metabolism in vivo in small intestine of fed rats. J Biol Chem 255:107-112, 1980 20. Wasserman DH, Lacy DB, Goldstein RE, et al: Exerciseinduced fall in insulin and hepatic carbohydrate metabolism during exercise. Am J Physiol256:E500-E508, 1989 21. Wasserman DH, Spalding JS, Lacy DB, et al: Glucagon is a primary controller of the increments in hepatic glycogenolysis and gluconeogenesis during muscular work. Am J Physiol 257:ElOSE117,1989 22. Geer RJ, Williams PE, Lairmore T, et al: Glucagon: an important stimulator of gut and hepatic glutamine metabolism. Surg Forum 38:27-29,1987 23. Wolfe RR, Goodenough RD, Wolfe MH, et al: Isotopic analysis of leucine and urea metabolism in exercising humans. J Appl Physiol52:458-466, 1982 24. Haralambie G, Senser L: Metabolic changes in man during long distance swimming. Eur J Appl Physiol43:115-125,198O 25. Refsum HE, Gjessing R, Stromme SB: Changes in plasma amino acid distribution and urine amino acids excretion during prolonged heavy exercise. Stand J Clin Lab Invest 39:407-413,1979 26. Wolfe RR: Measurement of urea kinetics in vivo by means of a constant tracer infusion of di-“N-urea. Am J Physiol 240:E428E434,1981 27. Carraro F, Hart1 W, Stuart C, et al: Whole-body and plasma protein synthesis in exercise and recovery in human subjects. Am J Physiol258:E821-E831,1990 28. Heitmann RN, Bergman EN: Transport of amino acids in whole blood and plasma of sheep. Am J Physiol 239:E242-E247, 1980 29. Chiasson JL, Liljenquist JE, Sinclair-Smith SC, et al: Gluconeogenesis from alanine in normal postabsorptive man. Diabetes 24~574-584,1975 30. Drewes LR, Conway WP, Gilboe DD: Net amino acid transport between plasma and erythrocytes and perfused dog brain. Am J Physiol233:E320-E325, 1977
of Gut and Liver in Nitrogen Richard J. Geer, Phillip E. Williams,
Metabolism
During
Exercise
Teresa Becker, D. Brooks Lacy, and Naji N. Abumrad
The role of the gut and liver in nitrogen metabolism was studied during rest, 150 minutes of moderate-intensity treadmill exercise, and 90 minutes of recovery in 18 hour-fasted dogs (n = 6). Dogs underwent surgery 16 days before an experiment for implantation of catheters in a carotid artery and in the portal and hepatic veins, and Doppler flow cuffs on the hepatic artery and portal vein. Arterial glutamine, alanine, and q-amino nitrogen (AAN) levels decreased gradually with exercise (P < .05), while arterial glutamate, NH,, and urea were unchanged. Net gut glutamine uptake was 1.3 2 0.5 pmol/kg . min at rest, and increased transiently to 2.5 2 0.3 +mol/kg min at 60 minutes of exercise (P < .05) as gut extraction increased. Net hepatic glutamine uptake was 0.6 2 0.4 kmol/kg min at rest, and increased to 3.4 f 0.6 and 2.6 + 0.5 kmol/kg . min after 60 and 150 minutes of exercise (P < .05) as hepatic extraction increased. Net gut glutamate and NH, output both increased transiently with exercise (P .: .05). These increases were matched by parallel increments in the net hepatic uptakes of these compounds. Alanine output by the gut and uptake by the liver were unchanged with exercise. Net gut AAN output was -2.1 ? 1.8 pmol/kg min at rest (uptake occurred), and increased transiently to 11.2 * 3.5 pmol/kg min after 30 minutes of exercise (P < .05). Net hepatic AAN uptake was 6.9 2 1.9 Fmol/kg . min at rest, and increased to 21.7 2 4.0 and 18.5 2 2.7 kmol/kg min after 30 and 150 minutes of exercise (P i .02). Net hepatic urea output increased from 3.8 2 0.8 pmol/kg min at rest to 6.8 ? 1.0 and 5.6 ? 1.0 pmol/kg min after 30 and 150 minutes of exercise (P < .05). In summary, during exercise, (1) glutamine uptake by the gut and liver is increased as fractional extraction rises; (2) gut amino acid and ammonia output occur in synchrony with, but exceed gut glutamine uptake; (3) hepatic glutamate and NH, uptake increase in parallel with the increased gut output of these substrates and appear to be closely regulated by their hepatic delivery rates; and (4) hepatic urea output is increased with the increase in hepatic uptake of nitrogenous precursors, but is quantitatively insufficient to explain the entire fate of these nitrogenous compounds within the liver. These studies demonstrate that gut and liver interactions play an important role in nitrogen metabolism during exercise. Copyright (il 199 1 by W. 6. Saunders Company
A
CCELERATED RATES of branched chain amino acid oxidation’.’ and adenosine monophosphate deaminatrot? lead to an increased production of ammonia in working muscle. The synthesis of glutamine’, and alanine’ appear to provide the chief vehicles by which excess ammonia can be shuttled away from this site. However, little is known about the subsequent metabolism of these amino acids during exercise, or the fate of the nitrogen that is liberated during the extramuscular breakdown of these amino acids. IJnder basal conditions, the splanchnic bed provides the primary site of glutamine and alanine removal.’ In the immediate postabsorptive state, glutamine is consumed by the gut and the carbon skeleton is oxidized or released into the portal vein as glutamate. The ammonia released in these reactions appears in the portal vein as such or is used in the synthesis of other amino acids. The liver is generally a net consumer of both alanine and glutamine,8,9 but under certain conditions glutamine may be released from this organ (eg, starvation,“’ acidosis”). When these amino acids are extracted by the liver, their carbon skeletons are either oxidized or used as precursors in gluconeogenesis and the nitrogen is utilized in ureagenesis or in the synthesis of amino acids and nucleotides.’ During exercise, the increased rate of muscle ammonia formation creates a greater importance for the avenues of glutamine and alanine disposal and nitrogen metabolism. The purpose of this study was to examine the magnitude and mechanism by which the gut and the liver interact in the metabolism of nitrogenous compounds during exercise. MATERIALS
Animals
and Sqicaf
AND METHODS
Procedures
Studies were performed on six mongrel dogs (mean weight, 21.4 t 1.0 kg) of either sex. that were maintained on a standard Merabolism,
Vol40,
No 3 (March).
1991:
pp 307-314
diet (Kal Kan beef dinner, Vernon, CA; and Wayne Lab Blox, St Louis, MO: 51% carbohydrate, 31% protein, 11% fat, and 796 fiber based on dry weight). Sixteen days before the experiment, a laparotomy was performed under general anesthesia (sodium pentobarbital, 25 mg/kg) and silastic sampling catheters (0.04 id) were inserted into the portal vein, the left common hepatic vein. and in a carotid artery. After their insertion, the catheters were filled with saline containing heparin (200 U/mL; Abbott Laboratories, North Chicago, IL) and their free ends were knotted. Doppler flow probes (obtained from Instrumentation Development Laboratory, Baylor University School of Medicine) were used to measure portal vein and hepatic artery blood flow.” Briefly. a small section of the portal vein, upstream from its junction with the gastroduodenal vein, was cleared of tissue, and the appropriate size flow cuff was placed around the vessel and secured. The gastroduodenal vein was isolated and then ligated proximal to its coalescence with the portal vein. Next, a section of the main hepatic artery lying proximal to the portal vein was isolated and the appropriate-size flow cuff was placed around the vessel and secured. The Doppler flow cuff leads and the knotted portal and hepatic vein catheter ends were placed in a subcutaneous pocket near where they exit from the abdominal cavity, and the knotted end of the carotid catheter was placed in a subcutaneous pocket in the ventral neck
From the Departments of Molecular Phwiolop and Blop!zysics. Surgery. and the Diabetes Research and Training Center, khnderbilt University School of Medicine, Nashville, TN. Supported by National Institutes of Health Grunt No. ROl DK42488, Diabetes Research and Training Grant No. SP60-AM-20593-08, and Clinical Nutrition Suppoti Unit Grant No. DK26657. D.H. W. ib a recipient of a Career Development Award fLom the Jwwwile Dlnbetrs Foundation International. Address reprint requests to David H. Wussemtan, PhD. Depaltmertt of Molecular Physiology and Bioph_vsks, Vanderbih (Jnitjersig School of Medicine, Nashville. TN 37232. Copyright c8 1991 by W.B. Saunders Compum 0026049519114003-0016$03.00l0 307
308
region. This procedure permitted complete closure of the skin incisions. Starting 1 week after surgery, dogs were conditioned to run on a motorized treadmill. The exercise duration and intensity were both increased progressively until a dog could perform the exercise parameters used in an experiment. Dogs were not exercised during the 48 hours preceding an experiment. Three days before each experiment, blood was withdrawn to determine the leukocyte count and the hematocrit of the animal. Only animals that had (1) a leukocyte count below 18,OOO/~L,(2) a hematocrit greater than 38%, (3) a good appetite (consuming all of the daily ration), and (4) normal stools, were used. On the day of the experiment, after an 18-hour fast, the free ends of the catheters were freed through a small skin incision made under local anesthesia (2% lidocaine, Astra Pharmaceutical, Worcester, MA). The contents of each catheter were aspirated, and the catheters were flushed with saline. Silastic tubing was connected to the exposed catheters and they were then brought over the back of the dog where they were secured with quick-drying glue allowing for convenient sampling. Saline was infused through the arterial catheter at a slow rate (0.1 mUmin) throughout the experiment. Experimental Procedures Experiments consisted of a control period (-40 to 0 minutes), a period of moderate-intensity (100 m/min, 12% grade) exercise (0 to 150 minutes), and an exercise recovery period (150 to 240 minutes. The work intensity utilized resulted in an increase in heart rate from 91 2 5 to 189 + 9 bpm. Indocyanine green was infused (0.1 mg/m’ min) starting 80 minutes before the control period and was continued for the duration of the experiment. This was used to obtain measurements of total hepatic blood flow to substantiate the determinations obtained with the Doppler flow cuffs, and to verify that the hepatic vein catheter was positioned correctly. Processing of Blood Samples Blood samples were collected from the carotid artery, portal vein, and hepatic vein in heparinized syringes and distributed into tubes containing EDTA (5 mg) or deproteinized in 4% perchloric acid (0.5 mL whole blood in 1.5 mL perchloric acid) and centrifuged. For the measurement of glutamine and glutamate, an aliquot of deproteinized blood from each sample was placed in each of two tubes containing 0.2 mol/L sodium acetate buffer. In one tube glutamine was completely reacted to glutamate with glutaminase (Sigma Pharmaceuticals, St Louis, MO). The amount of glutamate was then determined in both samples by measuring the conversion of NAD to NADH fluorometrically following the reaction catalyzed by glutamate dehydrogenase (Sigma Pharmaceuticals). The glutamine concentration was determined as the difference in glutamate concentrations between the tube that was reacted with glutaminase and the tube that was not. Blood n-amino nitrogen (AAN) levels were measured using o-phthaldehyde and measuring fluorescence in the presence of 2-mercaptoethanol.” Gomparisons in our laboratory show that values obtained using this method for the measurement of AAN levels agree within 5% to values obtained for total amino acids using a modification of the method of Heinrikson and Meridith.“’ Plasma alanine concentration was determined by reaction with ninhydrin. Plasma urea nitrogen was measured calorimetrically at 520 nm by a modification of the urea-diacetyl reaction.” Plasma ammonia levels were determined by the Berthiot reaction.16 Indocyanine green levels were determined spectrophotometrically (805 nm) in arterial and
WASSERMAN
ET AL
hepatic vein plasma samples immediately on completion of the study.
Calculations The net hepatic alanine, glutamine, glutamate, ammonia, and AAN balances were determined by the formula (4. A + P, P)H, H, where A is the arterial concentration, P the portal vein concentration, and H is the hepatic vein concentration. A,, P,, and H, are the arterial, portal vein, and total (A + P,) hepatic blood (plasma) flows as determined by Doppler flow probes. Plasma flow was determined as Blood Flow (1 - hematocrit). Splanchnic balances of these compounds were calculated as Hr. (A - H). The net hepatic balance of urea was calculated as H, H (A, A + P, . P). The hepatic fractional extraction of a compound equals its hepatic uptake divided by its rate of delivery to the liver (A. A + P, P). The net gut balance of a compound was calculated by the formula (A - P) . P,. The fractional extraction of a compound by the gut was calculated by dividing its uptake by the rate of delivery to the gut (P,. A). Glutamine, glutamate, urea, ammonia, and AAN balances were calculated using blood flow measurements and alanine balances were determined based on plasma flow rates. Statistical assessment of the basal versus exercise and recovery periods were performed initially using ANOVA for repeated measures. When the ANOVA yielded significant F values (P < .05), paired t tests were performed.” The statistics presented are the results of the post hoc t tests comparing basal with specific exercise and recovery values. Data are expressed as mean ? SE.
RESULTS
Glutamine Levels, Net Gut, Hepatic, and Splanchnic Balances, and Gut and Hepatic Fractional Extractions Arterial blood glutamine decreased gradually from levels of 665 I~I 31 kmol/L at rest to 557 ? 40 by the end of exercise (P < .05) (Fig 1). Portal and hepatic vein blood glutamine decreased initially with the onset of exercise from levels of 616 + 32 and 635 2 30 pmol/L to levels of 519 2 38 (P < ,005) and 489 + 48 (P < .Ol) pmol/L, respectively, after 60 minutes of muscular work (Fig 1). There were no further decreases in portal or hepatic vein blood glutamine after 60 minutes of exercise (Fig 1). Net gut glutamine uptake increased from a rate at rest of 1.4 2 0.5 to 2.5 ~fi:0.3 kmol/kg . min after 60 minutes of exercise (P < .05), and then declined throughout the remainder of the exercise period (Fig 1). The increase in net gut glutamine uptake was due to an increase in fractional extraction from a value of 0.08 -C 0.03 at rest to 0.16 * 0.02 after 60 minutes of exercise (P < .05) (Table 1). Net hepatic glutamine uptake increased from a rate of 0.6 ? 0.4 kmol/kg . min at rest to a peak of 3.4 ? 0.6 kmol/kg . min after 60 minutes of exercise (P < .05). This increase in uptake was due to an increase in hepatic fractional glutamine extraction from 0.03 * 0.02 at rest to a peak at 60 minutes of exercise of 0.20 + 0.05 (P < .02). Net splanchnic glutamine uptake increased immediately with the onset of exercise from a resting rate of 1.9 2 0.6 pmol/kg min to a peak of 5.9 + 0.7 pmol/kg . min at 60 minutes of exercise (P < .02) (Table 2).
EXERCISE
AND
SPLANCHNIC
1
NITROGEN
METABOLISM
309
A
EXERCISE
I 1
Glutamate Levels and Net Gut, Hepatic, and Splanchnic Balances and Hepatic Fractional Extraction
i
ARTERY
Arterial and hepatic vein blood glutamate levels were 42 t 7 and 44 ? 7 umol/L at rest and were essentially unchanged through exercise and recovery (Fig 2). Portal vein blood glutamate was 40 ? 6 umol/L at rest and increased transiently to a peak level of 56 _t 8 umol/L after 10 minutes of exercise (P < 0.05) (Fig 2). At rest, there was no net gut glutamate exchange (0.07 * 0.05 t.J,mol/kg min). Net gut glutamate output was evident transiently (-0.28 2 0.07 umol/kg . min at 30 minutes of exercise; P < .02). Net hepatic glutamate balance was not significantly different from 0 at rest (-0.09 t 0.08 umol/ kg. min), but transient hepatic glutamate uptake occurred with the onset of exercise (0.31 ? 0.12 kmol/kg min at 30 minutes; P < .05) (Tables 1 and 2).
B
Alanine Levels, Net Gut, Hepatic, and Splanchnic Balances. and Hepatic Fractional Extraction
-60
0
-30
30
60
90
120
150
180
210
Arterial plasma alanine levels were 346 f 20 kmol/L at rest and decreased to 238 2 13 umol/L by the end of exercise (P < .002) (Fig 3). Portal vein plasma alanine levels were 422 t 31 umol/L at rest, increased insignificantly with exercise, initially obtaining a level of 458 2 34 kmol/L at 30 minutes, after which levels decreased gradually to 292 2 18 FmoliL by the end of exercise (P < .Ol). Hepatic vein plasma alanine was 299 ? 20 pmol/L at rest and decreased to 101 +- 11 umol/L by the end of exercise (P < .002). The net gut balance of alanine was - 1.4 2 0.2 ).r,mol/kg . min at rest and -I .O 2 0.2 t.r,mol/kg . min at 150 minutes of exercise. Net hepatic alanine uptake was 2.5 + 0.3 kmolikg . min at rest and was essentially unchanged with exercise (3.1 2 0.4 p,mol/kg min after 30 minutes). Net hepatic fractional alanine extraction increased from 0.25 ? 0.02 at rest to 0.65 & 0.03 by the end of exercise (P < .OOl) (Table 1). Net splanchnic alanine uptake increased gradually from 1.1 2 0.1 to 2.1 I~I0.3 kmol/kg min (P < .05) (Table 2).
240
TIME (min)
AAN Levels, Net Gut, Hepatic, and Splanchnic Balances, Fig 1. (A) Blood arterial, portal vein, and hepatic vein glutamine concentrations and (B) net gut and (12) hepatic glutamine uptake during rest, 150 minutes of treadmill exercise, and 90 minutes of exercise recovery. Data are mean ‘_ SE; n = 6.
and Hepatic Fractional Extraction
Arterial, portal vein, and hcpatic vein AAN levels were 5,448 * 171, 5,375 t 213, and 5,179 t 192 ~mol/L at rest
Table 1. Gut and Hepatic Fractional Extraction
-
Exercise (time in minutes)
Gut fractional Glutamine Hepatic
-
30
10
Basal
Recovery (time in mfnutes)
90
60
150
120
240
180
160
extraction 0.08 2 0.03
fractional
0.13 + 0.02*
0.14 -C 0.03
0.16 t 0.02”
0.12 k 0.02
0.08 k 0.02
0.07 z 0.03
0.08 + 0.01
0.11 2 0.06
0.13 k 0.03
extraction
Glutamine
0.03 + 0.02
0.06 + 0.03
k 0.02*
0.16 t 0.04*
0.13 2 0.04*
0.06 + 0.03
0.10 2 0.02
0.06 2 0.07
0.12 k 0.08
0.16 k 0.03* 0.15 -c 0.06*
0.14
0.0
0.15 k 0.03* 0.20 + 0.07’
0.20 5 0.05’
Glutamate
0.05
2 0.09
Alanine
0.25 _f 0.02
0.29 -c 0.05
0.35 k 0.04*
0.38 k 0.06*
0.50 2 0.05’
0.59
-c 0.05*
0.04 + 0.08 0.65 k 0.03*
0.12 2 0.05 0.61 i 0.04*
0.07 k 0.04 0.60 k 0.04*
0.0 0.64 2 0.04*
Ammonia
0.39 + 0.08
0.47
k 0.10
0.50 2 O.ll*
0.46 2 0.09
+ 0.10
0.38
f 0.10
0.38
2 0.09
0.37
AAN
0.04 t 0.01
-
0.13 2 0.02’
-
-
0.13
-c 0.02*
NOTE.
Data are mean
*P < .05 compared
-c SE; n = 6. No net glutamate
with the basal state.
extraction
0.41
0.13 2 0.03’ was evident
in the basal period
+ 0.10
0.35 + 0.10
0.40 2 0.09
-
0.09 r 0.02*
0.05
or after 90 minutes
of exercise
recovery.
2 0.05
310
WASSERMAN
ET AL
Table 2. Net Splanchnic Balances Exercise Basal
Glutamine Glutamate
10
1.9 + 0.6 -0.02
30
3.6 f 0.8’
r 0.07
-0.01
(time
5.4 + 1.3*
90
5.9 2 0.7*
-
10.6?6.4
are in pmol/kg
Data are mean
min.
-
0.11 -c 0.03
0.10 f_ 0.03
0.11 t_ 0.02
2.11 2 0.27’
2.61 2 0.36’
2.50 + 0.51’
2.61 + 0.41*
Negative
numbers
3.6 f 0.6’ -0.06?
14.7 + 7.7 correspond
3.5 k 0.6’
17.2 + 8.7*
to splanchnic
output,
while
positive
numbers
7.1 + 6.8
10.0 ? 6.1
correspond
to splanchnic
-+ SE; n = 6. with the basal state.
/
*O r
0.12 + 0.03
1.87 + 0.26*
1.75 ? 0.24*
1.45 2 0.41
*P < .05 compared
0.12 + 0.03
0.13 2 0.03
0.16
1.43 ‘- 0.49
uptake.
3.8 + 0.6* 0.01 + 0.16
t 0.05
0.14 2 0.04
Balances
3.1 + 1.1
0.01 + 0.10
1.02 f 0.34
240
0.11 2 0.16
0.32 ? 0.10
0.15 z 0.04
in minutes)
160
0.14 2 0.16
3.2 + 0.5*
+ 0.07
1.07 * 0.12
(time
160
0.25
4.8 k l.l*
0.17
0.12 5 0.03
NOTE.
150
0.03 2 0.17
Alanine
9.0 f 2.7
Recovery 120
+ 0.13
Ammonia AAN
in minutes)
60
and levels in all vessels tended to decrease with exercise (Fig 4). However, levels decreased significantly with exercise only in the artery and hepatic vein (P < .05). Net gut AAN balance was not significantly different from 0 (2.1 2 2.8 kmol/kg . min), but AANs were produced during exercise (-11.2 ‘_ 3.5 kmol/kg . min at 30 minutes of exercise; P < .OS). Net hepatic AAN uptake was evident at rest (6.9 ? 1.9 pmol/kg . min) and increased with exercise (2117 * 4.0 and 18.5 t 2.7 pmol/kg . min after 30 and 150 minutes; P < .02). The increase in hepatic AAN uptake during exercise was due to a threefold increase in fractional extraction (0.04 ? 0.01 at rest and 0.13 * 0.02 after 1.50 minutes of exercise; P < .05) (Table 1). Net splanchnic
* I
VEIN
HEPATIC
A
EXERCISE
B
500
UPTAKE
I
HEPATIC
VEIN
II
L z
-0.4
’ -60
’
’
’
-30
0
30
60
90
TIME
’
’
120
150
180
’
’
210
240
(min) Fig 2. (A) Blood arterial, portal vein, and hepatic vein glutamate concentrations and (B) net gut and (C) hepatic glutamate balance during rest, 150 minutes of treadmill exercise, and 90 minutes of exercise recovery. Data are mean t SE; n = 6.
-61 -60
/ -30
0
30
60
’ 90
J 120
’
150
180
210
240
TIME (min)
Fig 3. (A) Plasma arterial, portal vein, and hepatic vein alanine concentrations and (B) net gut and hepatic alanine balance during rest, 150 minutes of treadmill exercise, and 90 minutes of exercise recovery. Data are mean + SE; n = 6.
EXERCISE AND SPLANCHNIC NITROGEN METABOLISM
311
A
EXERCISE
ammonia extraction was 0.39 + 0.08 at rest and increased with exercise to a peak of 0.50 * 0.11 at 30 minutes of exercise (P < .05) (Tables 1 and 2). Urea Levels and Net Hepatic Output
Plasma urea levels did not change significantly from rest through exercise in the artery, portal vein, and hepatic vein (Table 3). Arterial and portal plasma urea levels were similar from rest through exercise and recovery, indicating that the gut was not a net consumer or producer of this compound. Hepatic vein urea levels were higher in the hepatic vein than in the artery and portal vein throughout the experiment (Table 3). The urea concentration gradient across the liver (hepatic vein concentration minus arterial and portal vein concentrations) increased by twofold from 0.12 2 0.03 t.r,mol/L at rest to 0.23 * U.04 pmol/L at 30 minutes of exercise (P < .05). Net hepatic urea output increased from 3.6 ‘: 0.5 kmol/kg . min at rest to a peak rate of 6.8 t 0.7 kmol/kg min at 30 minutes of exercise (P < .05). Rates decreased gradually for the remainder of the exercise period (Fig 6). Hepatic Artery and Portal Vein Blood Flow I I I
1
I,, -:c
0
30
60
90
120
150
IPJO
210
240
Hepatic artery blood flow was 6.7 & 0.3 and 4.9 ? 0.6 mL/kg . min at rest and by the end of exercise, respectively
TIME (hl) (A) Blood arterial, portal vein, and rkpatic vein AAN concentrations and (B) net gut and hepatic AAN balances during rest, 150 minutes of treadmill exercise, and 90 minutes of exercise recovery. Data are mean rt SE; n = 6.
A
EXERCISE
8or 60 t
AAN uptake was evident at rest and increased gradually with exercise (Table 2). Differences between rest and exercise were significant only at 150 minutes of exercise. Anunonia Levels, Net Gut, Hepatic, and Splanchnic Baiances, and Hepatic Fractional Extraction
Arterial and hepatic vein plasma ammonia levels were 27 2 6 and 23 +- 5 p,mol/L at rest, respectively, and were unaffected by exercise (Fig 5). In contrast, portal vein plasma ammonia levels increased transiently from 40 ? 6 to 54 z 7 pmol/L after 30 minutes of exercise (P < .05). The gut released ammonia at rest (-0.32 + 0.09 kmol/kg min) and this release was accelerated transiently with exercise (-0.65 -c 0.21 Fmol/kg min at 30 minutes of exercise) (Fig 5). Similarly, net hepatic ammonia uptake was 0.46 ? 0.09 kmol/kg . min at rest and increased transiently with exercise (0.79 2 0.21 pmol/kg min at 30 minutes of exercise) (Fig 5). .4lthough gut output and hepatic uptake of ammonia increased with exercise in all dogs, in some animals the peak increments were evident after 10 minutes of exercise, while in others it was evident after 30 minutes. Hence, at no specific time point could significance be detected. However, when the basal period was compared with the transient increment, whether it occurred at 10 or 30 minutes, significance was obtained (P < .05). Net hepatic fractional
PTAk,E __bppI
_,
I -60
z
-30
f
I
0
30
-__
90
60
1:m
_.i
12c
I.>0
_ C’JTPUT
_-.-___-
_i
.50
?:O
240
i ,I !
Fig 5. (A) Plasma arterial, portal vein, and hepatic vein ammonia concentrations and (B) net gut and hepatic ammoniabalance during rest, 150 minutes of treadmill exercise, and 90 minutes of exercise recovery. Data are mean f SE: n = 6.
WASSERMAN
312
Table Exercise Basal
Arterial
10
3.58 2 0.49
3.55
r 0.49
3. Plasma (time
ET AL
Urea Levels Recoven/
in minutes)
30
60
90
3.63 t 0.49
3.65 f 0.48
3.72 2 0.49
120
3.76?
0.51
(time
in minutes)
150
160
180
240
3.58 2 0.54
3.84 -+ 0.53
3.84 t 0.53
3.88 i 0.53
Portal vein
3.60 2 0.49
3.56
r 0.49
3.61 -t 0.48
3.68
2 0.48
3.74 2 0.50
3.80 + 0.52
3.85 + 0.54
3.82 ? 0.54
3.86 + 0.53
3.87 + 0.53
Hepatictiein
3.72 i 0.52
3.75 f 0.51
3.84 2 0.51
3.87
f 0.51
3.94~
4.01
4.05 t 0.56
3.97 f 0.55
3.99 2 0.55
4.00 2 0.57
NOTE.
Levels are in mmol/L
and are expressed
as mean
DISCUSSION
Glutamine and alanine play important roles in the shuttling of nitrogen produced in the working muscle to the splanchnic bed. Glutamine is by far the more important nitrogen carrier, as its splanchnic uptake exceeded that for alanine by 78% at rest and by as much as 300% after 60 minutes of exercise. The importance of glutamine in nitrogen transport is especially apparent since this amino acid contains an amide group in addition to the AAN. The splanchnic uptake of AAN was well in excess of that for the sum of glutamine and alanine uptake, emphasizing the importance of other amino acids in the transfer of nitrogen to the splanchnic bed during rest and exercise. While alanine was consumed solely by the liver, the increased net splanchnic glutamine uptake during exercise was due to a transient twofold increase in net gut glutamine uptake,
EXERCISE
I I
I
z
I I 1
I
I
0 -60-30
I
I I I
I
w
I
0
30
60
90
+ 0.56
+ SE; n = 6.
(P < .05). No significant change in portal vein blood flow was evident with muscular work, as rates were 25.5 f 0.9 and 24.1 ? 2.1 mL/kg . min at rest and by the end of exercise, respectively (Table 4). The rates obtained with the Doppler flow probes yielded values for total hepatic blood flow that were within 10% of those obtained with indocyanine green during rest, exercise, and recovery (data not shown).
I
0.54
120150180210240
TIME (min) Fig 6. Net hepatic urse output during rest, 160 minutes of treadmill exercise, and 90 minutes of exercise recovery. Data are mean f SE; n = 6.
which peaked at 60 minutes of exercise, and to a more sustained increase in net hepatic glutamine uptake. Of the glutamine consumed by the splanchnic bed, approximately 27% was removed by the liver at rest, and approximately 57% and 72% were removed by the liver at 60 and 150 minutes of exercise. Hence, with the transition from rest to exercise, the balance of glutamine uptake by the splanchnic bed shifts from primarily gut to liver utilization. The percentage of the splanchnic glutamine uptake that could be attributed to the liver was reduced toward normal resting values during exercise recovery. The contribution of the liver was 50% of the total splanchnic glutamine uptake 90 minutes after the cessation of exercise. The data from this study indicate that the gut provides the liver with a source of carbon-based substrates for gluconeogenesis (glutamate, alanine, and other amino acids) and nitrogenous compounds (eg, ammonia and AAN) for ureagenesis at an increased rate during exercise. Since the peak increase in gut glutamine uptake coincides with the release of these hepatic substrates, it is likely to serve as a precursor for these compounds. In addition, to the role of gut glutamine metabolism in supplying substrate to the liver, glutamine oxidation may be an important source of energy for the gut.‘8,‘9Interestingly, the AAN output from the gut at 30 minutes of exercise exceeded the net gut uptake of glutamine by threefold. Moreover, the value obtained for net AAN output from the gut underestimates the absolute output due to the uptake of glutamine AAN (and likely other amino acids) by the gut. Hence, it is clear that gut proteolysis or the mobilization of a non-AAN source must be accelerated. Furthermore, these studies introduce the possibility that the gut is a labile substrate store that can be mobilized during exercise. The exercise-induced increase in net gut and hepatic glutamine uptakes occurred despite a decrease in the glutamine delivery to these organs. The entire increment in both net gut and hepatic glutamine uptake was due to an increase in the fractional extraction of this compound. Similarly, hepatic alanine and AAN uptakes were sustained despite decreases in their respective hepatic delivery rates as fractional extraction was increased. The exercise protocol used in this study elicits twofold increases in arterial plasma glucagon, norepinephrine, and epinephrine, and a 50% reduction in insulin.2”~2’Previous studies indicate that the increase in glucagon is essential to the exercise-induced increase in hepatic fractional alanine extraction.‘“” Furthermore, an increase in glucagon, similar to that which occurs with exercise, may lead to an increased extraction of glutamine by the gut and liver?’ In response to exercise, glutamate and particularly ammo-
313
EXERCISE AND SPLANCHNIC NITROGEN METABOLISM
Table 4. Hepatic Arteryand
Portal Vein Blood Flows Recovery (time in minutes)
Exercise (time in minutes) Basal Hepatic
artery
Portal vein
6.7
+
10
0.3
5.3 + 0.6*
25.5 + 1.4 24.3 t 1.5
NOTE. Blood flows are in mUkg lp
< .05 compared
30
5.4 f OS* 24.7 + 2.0
60
5.0 2 0.71 25.0 + 1.8
150
120
5.3 2 0.8
5.0 t 0.6*
25.0 & 2.4 24.2 + 2.3
5.0 t 0.6" 24.1 + 2.1
160
8.8 r 1.4
180
7.5 + 1.5
240
8.5 i-0.9
26.3 + 2.8 26.7 2 2.2 24.8 ? 1.3
min. Data are mean + SE; n = 6.
with the basal state.
were taken up by the liver with nearly the same dynamics as those that characterized the output of these compounds by the gut. Hence, while transient increases in portal vein glutamate and ammonia concentrations were evident within the initial 60 minutes of exercise, no changes in hepatic vein levels of these compounds were evident. Hence, the uptake of glutamate and ammonia were proportional to the rate at which they were delivered to the liver. Net hepatic urea output increased by almost twofold at 30 minutes of exercise to a rate of 6.8 pmol/kg . min. To our knowledge, this is the first direct report of an increase in hepatic urea output during exercise. The peak hepatic ourput of urea in the present study accompanied the peak in hepatic amino acid and ammonia uptakes. This finding is in contrast to work in humans that showed that stable isotope-determined urea production was unchanged over 10.5 minutes of exercise, despite a marked increase in protein breakdown.” Although there is no clear explanation for the discrepancy, it is possible that the relatively light work rate used by these investigators (30% of the maximum oxygen uptake) was insufficient to elicit a detectable increase in urea production. During competitive physical activity, an increase in urea production has been inferred from indirect indices (ie, changes in urea excretion, blood levels, and sweat losses).24.*5An alternative explanation for the apparent absence of an exerciseinduced increase in urea production may pertain to the large urea pool size, which may make isotope dilution methods somewhat insensitive, particularly under nonsteady-state conditions. Nevertheless, validation experiments in the resting, hepatectomized dog have been performed and indicate the measurement of urea turnover with stable isotopes is accurate under steady-state conditions and is generally within ~-20% in the non-steadystate.” Finally, we cannot rule out the possibility that species differences exist in hepatic metabolism, which permit a larger fraction of the nitrogen that is extracted by the liver to be channelled into urea in the dog compared with man. Although hepatic urea output was increased, the uptake of nitrogenous compounds by the liver was still far in excess of rhis rate. We could estimate by summing the hepatic uptakes of AAN, ammonia, and the glutamine amide group, that nitrogen uptake by the liver exceeded the rate at which nitrogen exited the liver via urea by twofold. The fate of the rest of the nitrogen that is extracted by the liver cannot be determined by the results of the present study. However, it has been proposed that hepatic uptake of nitrogenous compounds during exercise may be important for the accelerated formation of acute-phase proteins.”
nia
90
Despite the large increase in hepatic urea output in the present study, only a small gradual increase in arterial urea concentration was evident. This may be due to an increased urea disappearance rate during exercise, or to the large body pool that may effectively dampen the increase in this compound. The calculation of the organ balance of substrates measured in plasma requires assumptions regarding the equilibrium of these substrates between plasma and erythrocytes. Glutamine, glutamate, and AAN were measured in whole blood, and balances were calculated using blood flow: hence no assumptions pertaining to the equilibration across the erythrocyte were necessary. Alanine balances were calculated using plasma concentrations and flows. Similar alanine balances are generally obtained irrespective of whether the calculations are made using plasma or whole blood measurements.L8~Z9The assumption was made in the present study that urea and ammonia rapidly equilibrate across the erythrocyte membrane. Therefore, plasma levels were considered equal to whole blood levels and whole blood flow was used to calculate the gut and hepatic balance of these compounds. In support of this assumption is the report that arterial blood concentrations of both urea and ammonia are within 10% of levels in the plasma and the net balance of these compounds across the perfused dog brain is similar irrespective of whether blood or plasma values are used.‘” In conclusion, during exercise, (1) glutamine plays a greater role in nitrogen transfer from the periphery to the splanchnic bed than in the basal state; (2) gut and liver glutamine uptake is increased independently of load as fractional extraction is increased; (3) the gut AAN output exceeds the gut glutamine uptake, indicating that gut proteolysis may be accelerated; (4) hepatic glutamate and NH, uptakes increase in parallel with the increased gut output of these substrates and appear to be closely regulated by their hepatic delivery rates; (5) net hepatic urea output increases along with the hepatic uptake of nitrogenous precursors; and (6) quantitatively the hepatic uptake of nitrogenous compounds exceeds the output of urea, indicating that hepatic protein synthesis may be accelerated. The results of this study demonstrate the importance of gut and liver interactions in nitrogen metabolism during exercise.
ACKNOWLEDGMENT
The technical assistance of R. Ensley is gratefully acknowledged. The authors illustrations.
are grateful
to D.S. Wasserman
for preparation
of the
WASSERMAN
314
ET AL
REFERENCES
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