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

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

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 alpha -[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
Top
Abstract
Introduction
Methods
Results
Discussion
References

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.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

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.

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
R<SUB>a</SUB> = I<SUB>Leu</SUB> /EKIC<SUB>a</SUB> − I<SUB>Leu</SUB> (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
R<SUB>d</SUB> = R<SUB>a</SUB> − VdLeu<SUB>a</SUB> /d<IT>t</IT> (2)
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
NGLeuO = BF<SUB>pv</SUB> × (Leu<SUB>pv</SUB> − Leu<SUB>a</SUB> ) (3)
and
NLLeuO = BF<SUB>pv</SUB> × (Leu<SUB>hv</SUB> − Leu<SUB>pv</SUB> ) 
+ BF<SUB>ha</SUB> × (Leu<SUB>hv</SUB> − Leu<SUB>a</SUB>) (4)
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
GU<SUB>Leu</SUB> 
= (F<SUB>i*G</SUB> − F<SUB>o*G</SUB>)/[(Leu<SUB>ic*G</SUB> + KIC<SUB>ic*G</SUB>)/(Leu<SUB>icG</SUB> + KIC<SUB>icG</SUB>)] (5)
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
F<SUB>i*G</SUB> − F<SUB>o*G</SUB> = [(ELeu<SUB>a</SUB> × Leu<SUB>a</SUB> − ELeu<SUB>pv</SUB> × Leu<SUB>pv</SUB>)
+ (EKIC<SUB>a</SUB> × KIC<SUB>a</SUB> − EKIC<SUB>pv</SUB> × KIC<SUB>pv</SUB>)] × BF<SUB>pv</SUB> (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
LU<SUB>Leu</SUB> = (F<SUB>i*L</SUB> + F<SUB>i*L′</SUB> − F<SUB>o*L</SUB>)
× [(Leu<SUB>ic*L</SUB> + KIC<SUB>ic*L</SUB>)/(Leu<SUB>icL</SUB> + KIC<SUB>icL</SUB>)] (7)
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
F<SUB>i*L</SUB> + F<SUB>i*L′</SUB> − F<SUB>o*L</SUB> = [BF<SUB>pv</SUB> × (ELeu<SUB>pv</SUB> × Leu<SUB>pv</SUB> − ELeu<SUB>hv</SUB> 
× Leu<SUB>hv</SUB> + EKIC<SUB>pv</SUB> ×<SUB> </SUB>KIC<SUB>pv</SUB> − EKIC<SUB>hv</SUB> × KIC<SUB>hv</SUB>)] 
+ [Bf<SUB>ha</SUB> × (ELeu<SUB>a</SUB> × Leu<SUB>a</SUB> − ELeu<SUB>hv</SUB> × Leu<SUB>hv</SUB> + EKIC<SUB>a</SUB> 
× KIC<SUB>a</SUB> − EKIC<SUB>hv</SUB> × KIC<SUB>hv</SUB>)] (8)
The production of leucine by the gut (GPLeu) and liver (LPLeu) was calculated by the following equations
GP<SUB>Leu</SUB> = <IT>Eq. 5</IT> + F<SUB>oG</SUB> − F<SUB>iG</SUB> (9)
LP<SUB>Leu</SUB> = <IT>Eq. 7</IT> + F<SUB>oL</SUB> − F<SUB>iL</SUB> − F<SUB>IL′</SUB> (10)
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 alpha -[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.

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
% whole body proteolysis from the gut = <IT>Eq. 9/Eq. 1</IT> (11)
% whole body proteolysis from the liver = <IT>Eq. 10/Eq. 1</IT> (12)

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
Top
Abstract
Introduction
Methods
Results
Discussion
References

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

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).

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

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

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
Top
Abstract
Introduction
Methods
Results
Discussion
References

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 alpha -amino nitrogen was released from the gut at an increased rate during exercise (30). This was paralleled by an increase in net hepatic alpha -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 alpha -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.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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