Effect of physical activity and fasting on gut and liver
proteolysis in the dog
Amy E.
Halseth,
Paul J.
Flakoll,
Erica K.
Reed,
Allison B.
Messina,
Mahesh G.
Krishna,
D. Brooks
Lacy,
Phillip E.
Williams, and
David H.
Wasserman
Department of Molecular Physiology and Biophysics, Diabetes Research
and Training Center and Department of Surgery, Vanderbilt
University School of Medicine, Nashville, Tennessee 37232
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ABSTRACT |
The aim of this
study was to determine how gut and liver protein kinetics adapt to
acute exercise in the 18-h-fasted dog
(n = 7) and in dogs glycogen depleted
by a 42-h fast (n = 8). For this
purpose, sampling (artery and portal and hepatic veins) and infusion
(vena cava) catheters and Doppler flow probes (portal vein and hepatic
artery) were implanted with animals under general anesthesia. At least
16 days later, an experiment, consisting of a 120-min equilibration
period, a 30-min basal sampling period, and a 150-min exercise period,
was performed. At the start of the equilibration period, a constant
rate infusion of
[1-13C]leucine was
initiated. Gut and liver leucine appearance and disappearance rates
were calculated in these studies by combining a novel stable isotopic
method and arteriovenous difference methods. In the determination of
tissue leucine kinetics the tissue inflow of both
-[13C]ketoisocaproic
acid and [13C]leucine
was taken into account. The results of this study show that
1) the splanchnic bed (liver plus
gut) contributes ~40% to the whole body proteolytic rate in the
basal state and during exercise in dogs fasted for either 18 or 42 h,
2) the contributions of the gut and
liver to splanchnic bed proteolysis is about equal in the basal state
in both 18- and 42-h-fasted dogs, and
3) exercise in the 18-h-fasted dog
leads to a greater emphasis on gut proteolysis and a lesser emphasis on
hepatic proteolysis. These studies highlight the important contribution
of gut and hepatic proteolysis to whole body proteolysis and the
ability of the gut to acutely adapt to changes in physical activity.
gastrointestinal tract; ketoisocaproic acid; stable isotope; amino
acid
 |
INTRODUCTION |
THE SPLANCHNIC BED of postabsorptive humans releases
essential amino acids during exercise, showing that net protein
catabolism is occurring in these tissues (6). Studies in the dog, in
which hepatic and nonhepatic splanchnic tissue amino acid exchanges can
be measured, suggest that the gut is the source of the added amino
acids released in response to exercise (30). Furthermore, studies in
the dog model show that exercise increases the rate of entry of the
essential amino acid leucine into the portal vein (33), demonstrating
that the balance between gut protein synthesis and degradation is
shifted such that there is a greater emphasis on breakdown. An
important role of the gut in amino acid mobilization during exercise is
consistent with the demonstration in a variety of mammalian species
that the highest rates of protein turnover in the body are those in the
tissues of the splanchnic bed (8, 16-19, 31). Although the
capacity of the gut to rapidly mobilize leucine during exercise and
other metabolic stresses (13) is now known, there is still much that is
not understood. The factors, for example, that may influence splanchnic
proteolysis during exercise are unknown. Moreover, although the rates
of leucine exchange between the tissues of the splanchnic bed and blood
have been assessed, technical limitations have prevented the
quantitative association of leucine kinetics to protein kinetics. The
rate of entry of leucine into the blood underestimates tissue
proteolysis. This is because the intracellular leucine enrichment is
generally ~20-50% of the enrichment in the arterial plasma due
to the breakdown of protein in the cell. In the studies described in
this manuscript, ketoisocaproic acid (KIC) enrichment in the
blood draining hepatic and extrahepatic splanchnic tissues was
determined because KIC is in equilibrium with intracellular leucine
(12).
The experiments described herein were conducted to determine the
contribution of gut and liver to whole body proteolysis in the dog at
rest and during exercise in the immediate postabsorptive state (18-h
fast) and after a 24-h increase in fast duration (42-h fast). Compared
with 18-h-fasted dogs, 42-h-fasted dogs are ~80% glycogen depleted
and more reliant on the gluconeogenic pathway and the oxidation of
noncarbohydrate fuels (11). For these reasons protein stores may
potentially take on greater importance during fasting. We were
interested in determining whether the proteolytic response of
nonhepatic splanchnic tissue is greater in 42-h-fasted dogs in response
to exercise, under conditions in which amino acids are likely to be
more important in meeting the fuel needs of the animal, both as
gluconeogenic precursors for the liver and as oxidative substrates for
the working skeletal muscle. A stable isotopic method of assessing
regional proteolysis was used in combination with an arteriovenous
difference technique in a chronically catheterized dog model so that
protein kinetics could be determined in hepatic and nonhepatic
splanchnic tissues.
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METHODS |
Animal maintenance and surgical procedures.
Mongrel dogs (n = 15; mean weight 22.8 ± 0.3 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. The dogs were housed in a facility
that met American Association for the Accreditation of Laboratory
Animal Care guidelines, and the protocols were approved by Vanderbilt University's Institutional Animal Care and Use Committee. 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 for
tracer and indocyanine green infusions. 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 in the carotid artery. The carotid
artery was isolated, and a Silastic catheter (0.04 in. ID) was inserted
so that its tip rested in the aortic arch. After 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 (Instrumentation Development Laboratory, Baylor
University School of Medicine) were used to measure portal vein and
hepatic artery blood flows (10). Briefly, a small section of the portal
vein, upstream from its junction with the gastroduodenal vein, was
cleared of tissue, and a 7.0-mm ID 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. A section of
the main hepatic artery lying proximal to the portal vein was isolated
and a 3.0-mm ID flow cuff was placed around the vessel and secured. The
Doppler probe leads and the knotted free catheter ends, with the
exception of those of the carotid artery, 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 treadmill running. 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.
Dogs were studied in the postabsorptive state after an 18- or 42-h
fast. On the day of the experiment, the subcutaneous ends of the
catheters were freed through small skin incisions made with animals
under local anesthesia (2% lidocaine, Astra Pharmaceutical Products,
Worcester, MA) in the abdominal and neck regions. The contents of each
catheter were aspirated, and the catheters were flushed with saline.
Silastic tubing was connected to the exposed catheters and brought to
the back of the dog where they were secured with quick-drying glue.
Saline was infused in the arterial catheter throughout experiments (0.1 ml/min).
Experimental procedures.
As shown in Fig. 1, 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). The dogs were
subdivided into those in which the equilibration period had been
started after an 18-h fast (n = 7) and
those that had been studied after a 42-h fast
(n = 8). 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 (22).
After a baseline arterial blood sample was obtained, a primed, constant rate venous infusion of
[1-13C]leucine (14.4 µmol/kg; 0.24 µmol · kg
1 · min
1;
98% enriched) was initiated at t =
150 min, as was an infusion of indocyanine green (ICG; 0.1 mg · m
2 · min
1).
A primed, constant rate venous infusion of
[5-15N]glutamine was
also initiated at
150 min. Data from isotopic glutamine will be
presented in a separate report. Stable isotopes were
obtained from Cambridge Isotope Laboratories (Andover, MA), and ICG was
purchased from Hynson, Westcott, and Dunning (Baltimore, MD). ICG was
used as an independent backup measurement of splanchnic blood flow and
as a means of confirming hepatic vein catheter placement. We have
previously demonstrated good agreement between blood flow as determined
with Doppler flow probes and the ICG technique (9). 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 from the frequency shifts
of the pulsed sound signal emitted from the Doppler flow probes (10).

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Fig. 1.
Scheme illustrating protocol used in these studies in 18- (n = 7) and 42-h-fasted
(n = 8) dogs. All experiments
consisted of a 120-min equilibration period, a 30-min basal sampling
period, and 150-min moderate-intensity exercise period. A primed
infusion of leucine and an indocyanine green infusion were initiated at
t = 150 min and continued
throughout experiments. Arrows indicate when blood samples were taken
from artery, portal vein, and hepatic vein.
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Analysis of blood samples.
Blood samples were collected in EDTA and centrifuged at 2,000 g for 10 min at 4°C. The plasma
was transferred to separate tubes, which were stored on dry ice until
completion of the experiment. Samples were then stored at
70°C until later analysis. Internal standards, ketocaproate
and norleucine, were added to 1 ml of plasma, which was then
deproteinized with 1 ml of 8% perchloric acid. The supernatant was
passed over cation-exchange resin to separate the keto and amino acids.
The keto acids were further extracted according to the method of Nissen
et al. (24) using methylene chloride and 0.5 M ammonium hydroxide.
After evaporation under nitrogen gas, both the keto and amino acid
fractions were derivatized with
N-methyl-N-(t-butyldimethylsilyl)-trifluoroacetamide containing 1%
t-butyldimethylchlorosilane (Regis
Technologies, Morton Grove, IL) (28). Leucine enrichment and KIC
enrichment and concentration in the derivatized samples were determined
using gas chromatography-mass spectrometry (Hewlett-Packard 5890a GC and 5970 MS, San Fernando, CA). KIC and ketocaproate were detected at
both 301.0 and 302.0 atomic mass units (AMU), and leucine and norleucine were detected at both 302.2 and 303.2 AMU. Leucine concentrations were measured by the method described by Brown et al.
(2).
ICG was measured in plasma spectrophotometrically immediately after
experiments. Immunoreactive insulin was measured using a double
antibody system [interassay coefficient of variation (CV) of
10%] (21). Immunoreactive glucagon (3,500 mol wt) was measured
in plasma samples containing 50 µl of 500 kallikrein-inhibitor units/ml Trasylol (FBA Pharmaceuticals, New York, NY)
using a double antibody system (interassay CV of 7%) modified from the method developed by Morgan and Lazarow (21) for insulin. Glucagon and
insulin antiserums were obtained from Dr. R. L. Gingerich, Washington
University School of Medicine (St. Louis, MO), and the standard
glucagon and 125I-labeled glucagon
were obtained from Novo Research Institute (Copenhagen, Denmark).
Standard insulin and 125I-insulin
were obtained from Linco Research (St. Louis, MO). Plasma norepinephrine and epinephrine levels were determined with the use of a
high-performance liquid chromatography technique (interassay CVs of 11 and 13%, respectively) (20). Plasma cortisol was measured using the
Clinical Assays Gamma Coat Radioimmunoassay Kit (Clinical Assays,
Travenol-Genetech Diagnostics, Cambridge, MA) (interassay CV of 6%).
Calculations.
The equations below describe calculations for whole body, extrahepatic
splanchnic, and hepatic leucine kinetics using a combination of
isotopic and arteriovenous techniques. In the equations below and
throughout this report, the extrahepatic splanchnic tissue is referred
to simply as the gut. This is done for conciseness but is justified on
the basis that gastrointestinal tissue is the bulk of extrahepatic
splanchnic tissue based on mass and metabolic activity. This is
substantiated by the demonstration that the leucine enrichment in the
mesenteric vein and the portal vein is equal (33). Whole body rates of
appearance were calculated using the reciprocal pool model (27).
Because leucine is an essential amino acid, the entry of leucine into
the plasma can be used as an index of tissue proteolysis in the
postabsorptive state. For reasons described previously, arterial plasma
leucine enrichment is higher than in the cell and underestimates
proteolysis. For this reason, data from the keto acid of leucine, KIC,
was used in these calculations. KIC is formed by deamination of leucine inside the cell and is in rapid equilibrium with intracellular leucine.
The addition of unlabeled leucine to the intracellular pool, therefore,
is reflected by a decrease in the KIC enrichment, even if leucine is
oxidized without entering the plasma compartment. The equation for
whole body rate of appearance is described by Eq. 1
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(1)
|
Ra
is the rate of leucine appearance,
ILeu is the
[1-13C]leucine
infusion rate, and EKICa is the
arterial KIC enrichment. The rate of whole body leucine utilization,
Rd, was determined by the
following equation
|
(2)
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V
is the leucine volume of distribution and was assumed to be 150 ml/kg
and Leua is arterial plasma
leucine concentration. dLeua/dt
is the change in Leua with respect
to time. The theory behind
Eqs.
1 and 2 is described by Wolfe (34). Net gut
(NGLeuO) and hepatic (NLLeuO) leucine outputs were calculated by the
equations
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(3)
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and
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(4)
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where BFpv and
BFha are the portal vein and
hepatic artery blood flows, respectively, and Leu is the plasma leucine
concentration with the subscripts a, pv, and hv referring to artery,
portal vein, and hepatic vein samples, respectively.
Because leucine and KIC are in rapid equilibrium, leucine enrichment in
the tissue will be affected by the rates of
[13C]leucine and
[13C]KIC uptake. In
the model used to calculate tissue unidirectional fluxes here, KIC and
leucine are considered, in effect, a single pool, and total input and
output into this pool are calculated. Support for this is the
demonstration that KIC and leucine enrichments are the same in tissue
biopsies from a variety of tissues, including those of the splanchnic
bed (12). Also in support of the concept that leucine and KIC are in
equilibrium at the intracellular utilization site are the data in
Tables 2 and 3 that show that the enrichments of leucine and its keto
acid are nearly equivalent in the blood draining the gut and liver. The
diagram in Fig. 2 illustrates the model
that formed the basis of the calculations for unidirectional flux
through the tissues. The equations below describe the unidirectional flux through the tissue leucine (and KIC) pool and can be derived from
this scheme. For brevity and because the clear majority of the
flux goes through leucine, we will refer to these tissue fluxes as
tissue leucine production and utilization. Gut leucine utilization (GULeu) is the sum of gut
[13C]leucine and
[13C]KIC uptakes
divided by the intracellular enrichment of the leucine/KIC pool
|
(5)
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Leuic*G,
KICic*G,
LeuicG, and
KICicG are the intracellular
[13C]leucine,
[13C]KIC, leucine, and
KIC concentrations in extrahepatic splanchnic tissues. In practice,
EKIC in the portal vein, which drains the gut, is used to represent
intracellular enrichment in Eq.
5.
Fi*G
Fo*G is the inflow minus the
outflow of
[13C]leucine and
[13C]KIC and is
calculated as described in Eq.
6
|
(6)
|
ELeu refers to plasma leucine enrichment. Hepatic leucine
utilization (LULeu) was
determined by an equation analogous to that described in
Eq. 5
for the gut
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(7)
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Leuic*L,
KICic*L,
LeuicL and
KICicL are the intracellular
[13C]leucine,
[13C]KIC, leucine, and
KIC concentrations in the liver. Again, in practice, EKIC in the blood
draining the tissue (hepatic vein blood for the liver) is used to
represent intracellular leucine-KIC pool enrichment.
Fi*L + Fi*L'
Fo*L is the inflow minus the
outflow of
[13C]leucine and
[13C]KIC from the
liver and is calculated as described in
Eq.
8
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(8)
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The production of leucine by the gut
(GPLeu) and liver
(LPLeu) was calculated by the
following equations
|
(9)
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(10)
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FoG
FiG and
FoL
FiL
FIL' are the net outputs of
leucine and KIC for gut and liver, respectively, calculated based on
Eqs.
3 and 4.

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Fig. 2.
Schematic representation of model for calculation of hepatic and gut
proteolysis. There are 2 sources of
[13C]leucine in a
tissue: direct delivery of
[13C]leucine (Leu) by
circulation and that resulting from intracellular equilibration with
-[13C]ketoisocaproic
acid (KIC) delivered by circulation. For this reason, in calculation of
tissue unidirectional exchange, both rates of Leu and KIC uptake by
tissue bed were considered. * Stable isotopic
(13C) form of Leu or KIC
(throughout model). All values of F were calculated as blood flow in
vessel of interest multiplied by substrate concentration in that vessel
and have units of
µmol · kg 1 · min 1.
FiG, gut inflow of Leu + KIC;
FoG, gut efflux of Leu + KIC;
FiL, liver inflow of Leu + KIC
from portal vein; FiL',
liver inflow of Leu + KIC from hepatic artery;
FoL, liver efflux of Leu + KIC. GP
and LP are rates of Leu appearance from breakdown of protein in gut and
liver, respectively, with units of
µmol · kg 1 · min 1.
GU and LU are rates of irreversible loss of Leu + KIC from
intracellular pool of gut and liver, respectively.
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Whole body and tissue proteolysis was calculated from
Eqs.
1, 9,
and 10 by assuming that there are 590 µmol of leucine per gram of protein (31). The percent contributions
of the gut and liver to whole body proteolysis
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(11)
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(12)
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The experiments were designed so that they would consist of four
sampling periods. These are the 30-min basal period and the 25- to
50-min, 75- to 100-min, and 125- to 150-min exercise periods. Data were
pooled within each sampling period, and statistics were performed using
SuperAnova (Abacus Concepts, Berkeley, CA) on a MacIntosh PowerPC.
Statistical comparisons between groups and over time were made using
analysis of variance designed to account for repeated measures.
Specific time points were examined for significance using contrasts
solved by univariate repeated measures. Comparison between vessels was
made using a paired t-test. Statistics are reported in the corresponding table or figure legend for each variable. Differences were considered significant when
P values were <0.05. Data are
expressed as means ± SE.
 |
RESULTS |
Effects of 150 min of treadmill exercise in 18-h-fasted dogs.
Basal values for whole body, gut, and liver leucine kinetics are shown
in Table 1. Arterial leucine enrichment was
significantly greater than enrichments in either the portal or hepatic
veins throughout the experiment (P < 0.01; Table 2). Leucine enrichment fell in
response to exercise in all vessels (P < 0.05-0.01), but the fall was particularly marked in the portal
vein. Arterial KIC enrichment was equal in the artery, portal vein, and
hepatic vein in the basal state (Table 2). As was the case with leucine enrichment, KIC enrichment fell with exercise
(P < 0.05-0.01), and the fall
was greatest in the portal vein. The ratio of KIC to leucine enrichment
in the artery was lower than in the portal and hepatic veins throughout
the experiment (P < 0.01). In
contrast to the arterial blood, in which
EKICa was only 80% of
ELeua, the leucine enrichment was
equal to the KIC enrichment in the portal vein
(EKICpv/ELeupv = 1). This suggests that leucine and its ketoacid were in equilibrium
in the tissues of the gut (Table 2). The KIC enrichment in the hepatic
vein was actually 5-15% higher than the leucine enrichment in the
hepatic vein.
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Table 1.
Basal values for whole body, gut, and liver leucine kinetics and
percent contributions to whole body proteolysis of gut and
liver in 18- and 42-h-fasted dogs
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Table 2.
Arterial, portal vein, and hepatic leucine and KIC enrichments and
KIC-to-leucine enrichment ratios in basal state and during prolonged
exercise in 18-h-fasted dogs
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Arterial leucine levels were significantly reduced throughout the
exercise period (P < 0.01; Figs.
3B and
4A).
Whole body leucine output and proteolysis were significantly increased
from 75 to 100 min (P < 0.05; Fig.
4A and Table 4). Whole body leucine utilization was significantly increased from 25 to 50 min and from 75 to 100 min (P < 0.05; Fig.
4A). Net gut leucine output was
increased throughout the exercise period, indicating greater net
release (P < 0.05-0.01; Fig.
5A).
This increase was due to an increase in gut leucine production,
reflecting increased gut proteolysis, throughout the exercise period
(P < 0.05 to 0.01; Fig.
5A and Table 4). Gut utilization of
leucine was not significantly affected by exercise (Fig.
5A). Net hepatic leucine output was not significantly different from "zero" in the basal state and was not affected by exercise (Fig.
6A).
Hepatic leucine production was reduced by 30-40% in response to
exercise from 25 to 50 min and 75 to 100 min
(P < 0.05-0.02; Fig.
6A). Hepatic leucine utilization tended to decrease, but the change from basal was insignificant (Fig.
6A).

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Fig. 3.
Arterial plasma enrichments (A) and
concentrations (B) of Leu and KIC in
18- (n = 7) and 42-h-fasted
(n = 8) dogs. MPE, mole percent
excess. Data are means ± SE.
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Fig. 4.
Changes in arterial plasma Leu levels
(top) and whole body Leu production
and utilization (bottom) from basal
values in 18- (n = 7;
A) and 42-h-fasted
(n = 8;
B) dogs. Results are means ± SE
for 3 points in each interval (see
Calculations in
METHODS). * Significant change
from basal (P < 0.05-0.01).
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Fig. 5.
Changes in net gut leucine output
(top) and gut leucine production and
utilization (bottom) from basal
values in 18- (n = 7;
A) and 42-h-fasted
(n = 8;
B) dogs. Results are means ± SE
for 3 points in each interval (see
Calculations in
METHODS). * Significant
change from basal (P < 0.05-0.01).
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Fig. 6.
Changes in net hepatic leucine output
(top) and hepatic leucine production
and utilization (bottom) from basal
values in 18- (n = 7;
A) and 42-h-fasted
(n = 8;
B) dogs. Results are means ± SE
for 3 points in each interval (see
Calculations in
METHODS). * Significant change
from basal (P < 0.05-0.02).
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In the basal state, 17 ± 4% of the whole body proteolytic rate was
derived from the gut and 20 ± 4% was derived from the liver (Table
1). In response to exercise, there was a shift to a greater emphasis on
gut than liver proteolysis. From 125 to 150 min of exercise, 25 ± 4% of whole body proteolysis occurred in the gut, whereas 13 ± 4%
could be accounted for by hepatic proteolysis. The overall contribution
of the splanchnic bed to whole body protein breakdown was unaffected by
exercise, remaining at ~40%.
Arterial plasma hormone levels are shown in Table 5. Arterial plasma
insulin concentrations fell in response to exercise in the 18-h-fasted
dogs (P < 0.05 from
t = 75-100 min and 125-150 min). Arterial plasma glucagon (P < 0.05 from t = 75-100 min and 125-150 min), cortisol (P < 0.05-0.01 from t = 75-100
min and 125-150 min), norepinephrine
(P < 0.01 throughout the exercise period), and epinephrine (P < 0.01 throughout the exercise period) were increased. Portal vein blood flow
fell by ~15% throughout the exercise period
(P < 0.05-0.01), whereas
hepatic artery blood flow was unchanged (Table 6).
Effects of increasing the fast duration to 42 h on the basal state
and the responses to prolonged exercise.
Increasing the fast duration increased arterial leucine levels but did
not significantly alter the basal values for arterial leucine
enrichment, KIC enrichments, or whole body, gut, and liver leucine
kinetics (Table 1). Leucine enrichment was higher in the artery than in
the portal and hepatic veins throughout the experiment
(P < 0.01; Table
3). Leucine and KIC enrichments fell in
response to exercise. As was the case for the 18-h-fasted dogs, the
fall in enrichments was greatest in the portal vein. The ratio of KIC
to leucine enrichment was lower in the artery
(EKICa/ELeua ~0.80) than in the portal and hepatic veins. The ratios of KIC to
leucine enrichments in the portal and hepatic veins were generally ~1.00, indicating that leucine and KIC were in equilibrium in the
tissues of the gut and liver (Table 3).
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Table 3.
Arterial, portal vein, and hepatic leucine and KIC enrichments and KIC
to leucine enrichment ratios in basal state and during prolonged
exercise in 42-h-fasted dogs
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As was the case in 18-h-fasted dogs, exercise resulted in a decrease in
arterial leucine concentrations (P < 0.01 throughout the exercise period; Figs.
3B and
4B), a small increase in whole body
leucine production (P < 0.05-0.02 from 25 to 50 min and from 75 to 100 min), and a small
increase in whole body leucine utilization (P < 0.05-0.02 from 25 to 50 min and from 75 to 100 min; Fig. 4B). Net gut leucine output did not
change significantly in response to exercise (Fig.
5B). Gut leucine production,
however, was increased from 25 to 50 min and from 125 to 150 min of
exercise (P < 0.05), reflecting an
increase in gut proteolysis (Fig. 5B,
Table 4). Gut leucine utilization was
unchanged throughout the exercise period (Fig.
5B). Net hepatic leucine output was
unaltered by prolonged exercise, as was hepatic leucine production and
utilization (Fig. 6B). The percent
contributions of the gut and liver to whole body proteolysis were not
significantly affected by exercise in 42-h-fasted dogs.
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Table 4.
Whole body, gut, and liver proteolysis in basal state and during
prolonged exercise in 18- and 42-h-fasted dogs
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Of the arterial hormone measurements (Table
5), only basal cortisol was elevated in
42-h-fasted dogs compared with 18-h-fasted dogs. In response to
exercise, insulin fell (P < 0.05 from t = 125 to 150 min) and glucagon
(P < 0.05 throughout the exercise period), cortisol (P < 0.05-0.02 throughout the exercise period), norepinephrine
(P < 0.01 throughout the exercise
period), and epinephrine (P < 0.01 throughout the exercise period) rose. The early rise in cortisol
(P < 0.05 from
t = 25 to 50 min) and the increases in
norepinephrine and epinephrine in response to exercise were greater in
42-h-fasted dogs than in 18-h-fasted dogs. Portal vein blood flow fell
by ~15% throughout the exercise period
(P < 0.05-0.01), whereas
hepatic artery blood flow was unchanged (Table
6).
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Table 5.
Arterial plasma insulin, glucagon, cortisol, norepinephrine, and
epinephrine in 18- and 42-h-fasted dogs in basal state and during
prolonged moderate-intensity treadmill exercise
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Table 6.
Splanchnic blood flow in 18- and 42-h-fasted dogs in basal state and
during prolonged moderate-intensity treadmill exercise
|
|
 |
DISCUSSION |
The highest rates of protein turnover in the whole body are in the
tissues of the splanchnic bed (8, 16, 17, 18, 19, 31). It is
impossible, therefore, to understand the regulation of protein and
amino acid metabolism in vivo unless the control of splanchnic protein
metabolism is understood. Splanchnic protein synthesis can be
determined in vivo by assessing the rate of
incorporation of isotopic amino acids into protein in liver and tissues
of the gastrointestinal tract (18, 19, 23). Proteolysis of the splanchnic bed is more difficult to measure directly. In the present study, infusion of an isotope of the essential amino acid, leucine, and
arteriovenous difference methods were combined to assess gut and liver
proteolysis in the basal state and during a period of increased
physical activity. Splanchnic leucine kinetics were also studied during
the increased metabolic requirement of exercise when coupled to a
period of fuel deprivation that results in a near depletion of liver
and muscle glycogen. It is clear from these studies that, in the
sedentary 18-h-fasted dog, gut and liver proteolysis each comprise
15-25% of whole body proteolysis. The rate of gut proteolysis was
increased by ~40-50% at 125-150 min of exercise, and, as a
result, the percent contribution of the gut to whole body proteolysis
increased. This is consistent with our previous study that showed that
the rate of entry of leucine into arterial blood with exercise is due
to an increase in leucine entry into the portal vein (33). At the same
time the contribution of the liver to whole body proteolysis fell by ~7%. Although the rate that leucine was released into the hepatic vein declined during exercise in the previous study, the fall was
insignificant (33). Fuel deprivation, accomplished by fasting dogs for
42 h, did not lead to a greater increase in gut proteolysis in response
to exercise. The contribution of gut and liver proteolysis to whole
body proteolysis was not significantly altered from rest to the end of
exercise in 42-h-fasted dogs.
The finding in the present study that exercise does not increase
hepatic proteolysis is somewhat different from observations in the rat.
Rats exhibit a 10-15% fall in liver protein content after
prolonged exercise (>2 h), which is associated with an increase in
the activity of lysosomal proteolytic enzymes (15). The difference between these rat studies and the present studies in the dog may be due
to the relatively small drop in hepatic protein content observed in the
rat studies coupled with technical or species differences. The studies
in the rat are consistent with those in the dog in that an extended
fast did not alter the effect of exercise on liver protein breakdown
(15).
Whether working muscle becomes proteolytic during acute aerobic
exercise is controversial (25). The difference between total splanchnic
bed (gut + liver) proteolysis and whole body proteolysis during rest
and exercise in 18- and 42-h-fasted dogs changes very little. This
would then suggest that proteolysis by extrasplanchnic tissues changes
very little in response to exercise. Conversely, whereas whole body
leucine utilization was increased, gut and liver leucine utilization
was not. This suggests that skeletal muscle is the site of the
increased leucine utilization during exercise. This is consistent with
numerous other studies that indicate that exercise leads to an increase
in skeletal muscle leucine utilization (25). The primary metabolic fate
of the leucine consumed by working muscle is probably oxidation (32, 35). The fates of the leucine used by the gut and liver during exercise
have not been studied. In the resting, overnight-fasted dogs only ~5
and 15% of the leucine used by gut and liver, respectively, is
oxidized (36), which is in agreement with data reported in sheep (16).
The remainder then is primarily incorporated into protein.
To improve the accuracy of leucine kinetic measurements and to relate
these measurements more precisely to proteolysis, a novel application
of an isotopic technique to the arteriovenous difference method was
used. This method considers the rapid equilibration of leucine and KIC
inside the cell (12, 27, 34). The approach used in the present study is
unique in that it accounts for both the
[13C]leucine and
[13C]KIC entry into
tissue in the calculation. This resulted in values for tissue
proteolysis that were ~20% higher than those derived when only
tissue [13C]leucine
uptake was used (data not shown). The approach used in the present
study also takes the rapid equilibrium of leucine and KIC into account
by using the KIC enrichment in the venous blood draining the gut and
the liver to represent the intracellular enrichment of leucine. Tables
2 and 3 provide evidence for the rapid equilibrium of leucine and its
keto acid. Although the leucine enrichment is higher than the KIC
enrichment in arterial blood entering the splanchnic bed, the
enrichments in the venous blood that exit these tissues are nearly
equal. This finding is consistent with an earlier report that showed
that, during an infusion of [14C]leucine, arterial
leucine specific activity was 30% higher than arterial KIC specific
activity, but portal vein leucine and KIC specific activities were
equal (1). One corollary of this is that it really matters very little
whether the venous leucine or venous KIC enrichment is used for the
calculation of gut and liver leucine kinetics, since they are nearly
equal. These results also provide support for the use of the reciprocal
pool model described by Schwenk and colleagues (27) for calculation of whole body leucine kinetics. This model uses arterial or mixed venous
KIC enrichment to reflect intracellular leucine enrichment. Results of
the present study show that the arterial KIC enrichment is very close
to the KIC and leucine enrichments in the venous blood draining gut and
liver. Thus the arterial KIC enrichment reflects leucine enrichment in
the tissues of the splanchnic bed, the site of ~40% of whole body
proteolysis.
It was somewhat surprising that the extended fast did not significantly
increase whole body or splanchnic proteolysis or decrease leucine
uptake by the splanchnic tissues. Fasting has been shown to decrease
protein mass in rat liver (19), small intestine (19, 26), colon (26),
and stomach (26). This is due, at least in part, to a decrease in
fractional protein synthetic rate. The mRNA levels for enzymes in the
small intestine and colon that catalyze proteolysis are increased with
fasting in the rat (26). If these changes in mRNA correspond to changes
in protein and enzyme level, proteolysis would also be expected to be
higher. The reason that the 42-h-fasted dogs do not show a decrease in leucine utilization (an index of protein synthesis) and an increase in
proteolysis is partly because dogs have a slower transition to the
fasted state based on liver glycogen levels (11) and gastrointestinal
absorption profiles (5). A 42-h-fasted dog is probably not the
metabolic equivalent of the rat fasted for a similar duration. The
adaptations to fasting are dynamic and depend on the length of food
deprivation (26). It is possible that a longer fast might lead to a
detectable increase in proteolytic rate. It is also feasible that
differences between these studies and those using tissue biopsies in
the rat may be a result of the different techniques used (19, 26).
Although the methodology used in this study is more sensitive at
detecting acute changes in protein kinetics and has the advantage of
allowing for serial samples in the same animal, the biopsy
technique may provide a larger signal for detecting the cumulative
changes that occur over an extended interval.
The signal or signals that lead to the increase in gut proteolysis in
response have yet to be defined. The gut is exposed to increased
sympathetic drive (4) and a number of hormonal changes during exercise
(7). Fasting is known to increase the insulin counterregulatory hormone
response to exercise and to decrease insulin levels (7). We initially
speculated that the greater adrenergic drive or cortisol response that
occurs with exercise after a prolonged fast might increase the
proteolytic response. This was not the case. It is possible that
catecholamines or cortisol causes the increment in proteolysis observed
with exercise, but the gut is insensitive to the differences observed between 18- and 42-h-fasted exercising dogs. In humans, an increase in
corticosterone (the predominant glucocorticoid in humans) has been
demonstrated to alter whole body proteolysis; however, this effect was
not observed until ~4 h of administration (29). Therefore, it is
possible that the glucocorticoid effect may be too slow to result in
changes in proteolysis during an acute exercise bout. The fall in
insulin may also conceivably facilitate gut proteolysis. However,
suppression of insulin levels in resting dogs with the use of
somatostatin does not affect proteolysis, at least not after an
overnight fast (14). In short, it is clear that there is not a
correlation between the magnitude of the exercise-induced change in any
of the hormones measured and the exercise-induced increase in gut
proteolysis.
The functional significance of the increase in gut proteolysis during
exercise probably relates, in part, to the ability of the splanchnic
tissue to supply carbons for the gluconeogenic pathway and amino acids
to the protein synthetic pathways of the liver. An earlier study showed
that
-amino nitrogen was released from the gut at an increased rate
during exercise (30). This was paralleled by an increase in net hepatic
-amino nitrogen uptake, which was more than adequate to account for
the disposal of amino acids released by the gut. Some of the amino
acids are used for gluconeogenesis, whereas branched-chain amino acids
appear, for the most part, to escape hepatic extraction and are used by skeletal muscle (6). It could be estimated by subtracting net hepatic
urea output from net hepatic
-amino nitrogen uptake that a
considerable quantity of nitrogen is retained in the liver, suggesting
that amino acids may not be degraded but may be used for hepatic
protein synthesis (3). Because amino acids released due to gut
proteolysis enter the portal vein, which directly perfuses the liver, a
second physiologically significant aspect of the increase in gut
proteolysis is the possibility that amino acids delivered via the
portal vein are preferentially metabolized compared with amino acids
via the hepatic artery. This concept is consistent with a previous
report that showed that all the leucine used to synthesize fibrinogen
in the liver is derived from the portal vein (1).
In summary, regional proteolysis was calculated in these studies using
a novel application of the reciprocal pool model for measuring leucine
kinetics. With this approach, the tissue inflow of
[13C]KIC and
[13C]leucine are both
taken into account and KIC enrichment in venous blood is used to
represent the intracellular enrichment of the KIC-leucine pool. The
present study is consistent with earlier work that showed that the gut
contributes leucine to the circulating leucine pool at an increased
rate during exercise (33). In addition, the methodology used in this
study allowed the intracellular isotope dilution of the ratio of
[13C]leucine to
[13C]KIC to be
measured and tissue proteolysis to be calculated. When regional
proteolytic rates are then compared with whole body proteolysis, the
contribution of the splanchnic bed (liver plus gut) can be shown to be
~40% of the total in the basal state and during exercise regardless
of the degree of fasting. The individual contributions of the gut and
liver to splanchnic bed proteolysis were about equal in the basal state
in 18- and 42-h-fasted dogs. Exercise in the 18-h-fasted dog, but not
in dogs fasted for 42 h, led to a greater emphasis on gut proteolysis
and a lesser emphasis on hepatic proteolysis. In conclusion, these
studies demonstrate the important role of gut and hepatic proteolysis
to the whole body response and the ability of the gut to adapt to
changes in physical activity.
 |
ACKNOWLEDGEMENTS |
The authors are grateful to Li Zheng, Wanda Snead, Pamela Venson,
Eric Allen, and Thomas Becker for excellent technical assistance.
 |
FOOTNOTES |
This work was supported by National Institute of Diabetes and Digestive
and Kidney Diseases Grant RO1 DK-47344 and Diabetes Research and
Training Center Grant 5 P60 DK-20593.
Address for reprint requests: D. H. Wasserman, Light Hall Rm. #702,
Dept. of Molecular Physiology and Biophysics, Vanderbilt Univ. School
of Medicine, Nashville, TN 37232-0615.
Received 19 February 1997; accepted in final form 8 August 1997.
 |
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