Glucagon response to exercise is critical for accelerated hepatic glutamine metabolism and nitrogen disposal

Mahesh G. Krishna, Robert H. Coker, D. Brooks Lacy, Bradley A. Zinker, Amy E. Halseth, and David H. Wasserman

Department of Molecular Physiology and Biophysics and Diabetes Research and Training Center, Vanderbilt University School of Medicine, Nashville, Tennessee 37232


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The aim of this study was to determine the role of glucagon in hepatic glutamine (Gln) metabolism during exercise. Sampling (artery, portal vein, and hepatic vein) and infusion (vena cava) catheters and flow probes (portal vein, hepatic artery) were implanted in anesthetized dogs. At least 16 days after surgery, an experiment, consisting of a 120-min equilibration period, a 30-min basal sampling period, and a 150-min exercise period, was performed in these animals. [5-15N]Gln was infused throughout experiments to measure gut and liver Gln kinetics and the incorporation of Gln amide nitrogen into urea. Somatostatin was infused throughout the study. Glucagon was infused at a basal rate until the beginning of exercise, when the rate was either 1) gradually increased to simulate the glucagon response to exercise (n = 5) or 2) unchanged to maintain basal glucagon (n = 5). Insulin was infused during the equilibration and basal periods at rates designed to achieve stable euglycemia. The insulin infusion was reduced in both protocols to simulate the exercise-induced insulin decrement. These studies show that the exercise-induced increase in glucagon is 1) essential for the increase in hepatic Gln uptake and fractional extraction, 2) required for the full increment in ureagenesis, 3) required for the specific transfer of the Gln amide nitrogen to urea, and 4) unrelated to the increase in gut fractional Gln extraction. These data show, by use of the physiological perturbation of exercise, that glucagon is a physiological regulator of hepatic Gln metabolism in vivo.

nitrogen; dog; exertion; liver; amino acid


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

GLUTAMINE (Gln), the most abundant amino acid in the body, is not only a substrate for gluconeogenesis but also a major vehicle for nitrogen delivery. The importance of hepatic Gln metabolism is exemplified by studies in the exercising dog, during which hepatic Gln uptake increases by twofold because of an increase in hepatic fractional extraction (8). Moreover, exercise causes an ~80% increase in the conversion of Gln to urea, because essentially all of the amide nitrogen on the Gln taken up by the liver goes to the formation of urea (8).

Despite the important role of this amino acid in nitrogen metabolism, very little is known about how hepatic Gln metabolism is regulated in vivo. The hypothesis tested in the present study is that the increase in glucagon is the signal that determines the exercise-induced increase in hepatic Gln uptake and incorporation of the amide nitrogen to urea. This is based on the demonstration that glucagon acutely stimulates net Gln uptake and flux through the glutaminase step in the isolated, perfused rat liver (13), increases glutaminase activity in hepatocytes (3), and rapidly stimulates the formation of [15N]urea from [15N]Gln (21). A role for the rise in glucagon in the exercise-induced increase in the net hepatic fractional extraction of alanine has already been shown (26).

In the present study, the role of the increase in circulating glucagon during exercise was tested in the chronically catheterized dog. Somatostatin was infused intravenously to suppress endogenous pancreatic hormone release, and insulin and glucagon were replaced at the rates necessary to mimic or prevent the increase in glucagon secretion normally seen during exercise. Hepatic nitrogen metabolism was quantified using stable isotopic and arteriovenous difference methods.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animal maintenance and surgical procedures. Mongrel dogs (n = 10; mean wt 24.5 ± 0.8 kg) of either gender that had been fed a standard diet (Kal Kan beef dinner, Vernon, CA and Wayne Lab Blox: 51% carbohydrate, 31% protein, 11% fat, and 7% fiber based on dry weight, Allied Mills, Chicago, IL) were studied. Dogs were fed once daily. The total energy intake of this diet corresponded to 85 kcal · kg-1 · day-1, and the absolute protein intake was 6 kcal · kg-1 · day-1. In dogs maintained on this diet, body weight is unchanged from the day of surgery to the day of the experiment. The dogs were housed in a facility that met American Association for Accreditation of Laboratory Animal Care guidelines, and the protocols were approved by Vanderbilt University's Institutional Animal Care and Use Subcommittee.

At least 16 days before each experiment, a laparotomy was performed under general anesthesia (0.04 mg/kg of atropine and 15 mg/kg pentothal sodium presurgery and 1.0% isoflurane inhalation anesthetic during surgery). Silastic catheters (0.03 in ID) were inserted into the vena cava and splenic and mesenteric veins for infusions. The tips of the splenic and mesenteric vein infusion catheters were advanced just beyond where they coalesce with the portal vein. Silastic catheters (0.04 in ID) were inserted into the portal vein and left common hepatic vein for blood sampling. Incisions were also made in the neck region for the placement of a sampling catheter (0.04 in ID) in the carotid artery. The carotid artery catheter was advanced so that its tip rested in the aortic arch. After insertion, the catheters were filled with saline containing heparin (200 U/ml), and their free ends were knotted. Doppler flow probes (Instrumentation Development Laboratory, Baylor University School of Medicine) were used to measure portal vein and hepatic artery blood flows, as described previously (8, 12, 14). The Doppler probe leads and the knotted free catheter ends, with the exception of the knotted end of the carotid artery catheter, were stored in a subcutaneous pocket in the abdominal region so that complete closure of the skin incision was possible. The free end of the carotid artery catheter was stored under the skin of the neck.

Starting 1 wk after surgery, dogs were exercised on a motorized treadmill so that they would be familiar with the procedures. Animals were not exercised during the 48 h preceding an experiment. Only animals that had 1) a leukocyte count >18,000/mm3, 2) a hematocrit >36%, 3) normal stools, and 4) a good appetite (consuming all of the daily ration) were used.

On the day of the experiment, with the use of local anesthesia (2% lidocaine), the subcutaneous ends of the catheters were freed through small skin incisions made on the subcutaneous pockets in which catheters were stored. The contents of each catheter were aspirated, and catheters were flushed with saline. Silastic tubing was connected to the exposed catheters and brought to the back of the dog, where the tubing was secured with quick-drying glue. Saline was infused in the arterial catheter throughout the experiments (0.1 ml/min).

Experimental procedures. Experiments were performed in 18-h-fasted dogs. All experiments consisted of a 120-min equilibration period (-150 to -30 min), a 30-min basal sampling period (-30 to 0 min), and a 150-min moderate-intensity exercise period (0 to 150 min). Exercise was performed at 100 m/min, 12% grade on a motorized treadmill. The exercise intensity used in these experiments has previously been shown to result in a twofold increase in heart rate and an increase in O2 uptake to 50% of maximum (20). After a baseline arterial blood sample was obtained, a primed constant-rate venous infusion of [5-15N]Gln (36 µmol/kg prime, 0.4 µmol · kg-1 · min-1 infusion) was initiated at t = -150 min. [5-15N]Gln was obtained from Cambridge Isotope Laboratories (Andover, MA).

Somatostatin was infused into the vena cava beginning at t = -150 min and was continued until the end of the study (0.8 µg · kg-1 · min-1). Glucagon and insulin were infused into the portal vein also, beginning at t = -150 min. Glucagon was infused at a basal rate (0.65 ng · kg-1 · min-1) until the beginning of exercise (0 min), at which time the rate was either 1) gradually increased to simulate the glucagon response to this exercise protocol (25) (SimG; n = 5) or 2) unchanged to maintain glucagon levels at basal (BasG; n = 5). Insulin was infused intraportally at 250 µU · kg-1 · min-1 beginning at t = -150 min. The insulin infusion rate was then adjusted during the equilibration period, on the basis of feedback from glucose values obtained by rapid arterial sampling, to achieve stable euglycemia. The insulin infusion rate at which this stable glucose level was obtained was 230 ± 33 µU · kg-1 · min-1 in SimG and 230 ± 25 µU · kg-1 · min-1 in BasG. As described previously (25), the insulin infusion rate was adjusted during exercise to simulate the normal exercise-induced fall in insulin in both groups. Arterial glucose was clamped during the exercise periods using a glucose infusion the rate of which was based on feedback from small arterial samples. Arterial, portal vein, and hepatic vein samples were drawn at t = -30, -15, 0, 25, 37.5, 50, 75, 87.5, 100, 125, 137.5, and 150 min. Portal vein and hepatic artery blood flows were recorded continuously on-line (12).

Processing of blood and tissue samples. Blood samples were collected in heparinized syringes and put in tubes containing 1) EDTA for analysis of plasma, 2) 4% perchloric acid for analysis of whole blood, or 3) EGTA and glutathione for analysis of plasma catecholamines. Samples were then centrifuged, and the supernatant was removed. Gln (18), glutamate (Glu) (18), and urea (17) concentrations were measured in the supernatant from perchloric acid-deproteinized blood. Plasma glucose concentration was measured during the experiments with the glucose oxidase method. Plasma Gln enrichment, expressed as the molar percent excess, was determined using the tertiary butyldimethylsilyl derivatives. Derivatized samples were analyzed on a Hewlett-Packard 5957 gas chromatography-mass spectrometry system by use of electron ionization and selected ion monitoring of ions with mass-to-charge ratios of 431/432 for Gln. Plasma urea enrichment was measured by Metabolic Solutions (Merrimack, NH). For this purpose, samples were reacted with urease in a sealed tube in which a trapping well containing H2SO4 was placed. The NH4 that was formed was released from solution with an alkalinizing agent (5 M NaOH) and trapped in the wells, forming ammonium sulfate. The N enrichment of an aliquot of the ammonium sulfate solution was analyzed using isotope ratio mass spectrometry. Plasma insulin, glucagon, cortisol, and catecholamine analyses were done by established techniques that have been described previously (10).

Calculations. Gln appearance (Ra) in and disappearance (Rd) from arterial plasma were measured using an isotope dilution method described by Eqs. 1 and 2, respectively. The validity of this method has been recently assessed for the Gln system (16).
R<SUB>a</SUB><IT>=</IT>(R*/E<SUB>a</SUB>)<IT>−</IT>R*<IT>−</IT>p<IT>×</IT>V<IT>×</IT>[Gln]<SUB>a</SUB><IT>×</IT>dE<SUB>a</SUB>/(d<IT>t×</IT>E<SUB>a</SUB>) (1)

R<SUB>d</SUB><IT>=</IT>R<SUB>a</SUB><IT>−</IT>p<IT>×</IT>V<IT>×</IT>d[Gln]<SUB>a</SUB>/d<IT>t</IT> (2)
R* is the infusion rate of [5-15N]Gln, Ea is the arterial enrichment of Gln, [Gln] refers to the blood Gln concentrations, the subscript a is used to designate arterial blood, V is the volume of Gln distribution, and p is the pool fraction. V and p are assumed to be 360 ml/kg and 0.75, respectively, on the basis of work by Kreider et al. (16).

Extrahepatic splanchnic balance is calculated as the difference between arterial and portal venous blood multiplied by portal venous flow. For brevity, and because most of this tissue bed is gut, we have defined extrahepatic splanchnic balances as gut balances. Net gut and liver Gln balance (NGGlnB and NHGlnB, respectively) were calculated as follows
NGGlnB<IT>=</IT>([Gln]<SUB>a</SUB><IT>−</IT>[Gln]<SUB>pv</SUB>)<IT>×</IT>BF<SUB>pv</SUB> (3)

NHGlnB<IT>=</IT>[Gln]<SUB>pv</SUB><IT>×</IT>BF<SUB>pv</SUB><IT>+</IT>[Gln]<SUB>a</SUB>

×BF<SUB>ha</SUB><IT>−</IT>[Gln]<SUB>hv</SUB><IT>×</IT>(BF<SUB>pv</SUB><IT>+</IT>BF<SUB>ha</SUB>) (4)
where the subcripts pv, ha, and hv refer to portal vein, hepatic artery, and hepatic vein, respectively, and BF refers to blood flow. Net Glu balances were calculated in a similar manner. Net hepatic urea output was calculated by an equation analogous to Eq. 4, except that the + and - signs were reversed so that positive numbers would reflect net output. Unidirectional gut (GGlnFE) and hepatic (HGlnFE) fractional Gln extraction was calculated using isotopic Gln, such that
GGlnFE<IT>=</IT>(E<SUB>a</SUB><IT>×</IT>[Gln]<SUB>a</SUB><IT>−</IT>E<SUB>pv</SUB><IT>×</IT>[Gln]<SUB>pv</SUB>)<IT>/</IT>(E<SUB>a</SUB><IT>×</IT>[Gln]<SUB>a</SUB>) (5)

HGlnFE<IT>=</IT>{(E<SUB>pv</SUB><IT>×</IT>[Gln]<SUB>pv</SUB><IT>×</IT>BF<SUB>pv</SUB>)<IT>+</IT>(E<SUB>a</SUB><IT>×</IT>[Gln]<SUB>a</SUB><IT>×</IT>BF<SUB>ha</SUB>)

−(E<SUB>hv</SUB><IT>×</IT>[Gln]<SUB>hv</SUB>)<IT>×</IT>(BF<SUB>pv</SUB><IT>×</IT>BF<SUB>hv</SUB>)}<IT>/</IT>(E<SUB>pv</SUB><IT>×</IT>[Gln]<SUB>pv</SUB> (6)

<IT>×</IT>BF<SUB>pv</SUB><IT>+</IT>E<SUB>a</SUB><IT>×</IT>[Gln]<SUB>a</SUB><IT>×</IT>BF<SUB>ha</SUB>)
Gln delivery to gut and liver (GGlnD and HGlnD, respectively) were calculated by the following equations
GGlnD<IT>=</IT>[Gln]<SUB>a</SUB><IT>×</IT>BF<SUB>pv</SUB> (7)

HGlnD<IT>=</IT>[Gln]<SUB>a</SUB><IT>×</IT>BF<SUB>ha</SUB><IT>+</IT>[Gln]<SUB>pv</SUB><IT>×</IT>BF<SUB>pv</SUB> (8)
Unidirectional gut (GGlnU) and hepatic (HGlnU) Gln uptake from the blood could then be calculated from the equations
GGlnU<IT>=</IT>GGlnFE<IT>×</IT>GGlnD (9)

HGlnU<IT>=</IT>HGlnFE<IT>×</IT>HGlnD (10)
and unidirectional gut (GGlnO) and hepatic (HGlnO) Gln output into the blood could be calculated as
GGlnO<IT>=</IT>GGlnU<IT>−</IT>NGGlnB (11)

HGlnO<IT>=</IT>HGlnU<IT>−</IT>NHGlnB (12)
The rate of conversion of the Gln amide nitrogen into urea was calculated as
conversion of Gln amide nitrogen into urea

=<FENCE>&Dgr;Urea E<SUB>a</SUB>(<IT>150 </IT>min<IT>−0 </IT>min)<FENCE><LIM><OP>∫</OP><LL><IT>0</IT></LL><UL><IT>t</IT></UL></LIM></FENCE> GlnE<SUB>hv</SUB></FENCE> (13)

(from<IT> t=0 </IT>to 150 min)]<IT>×</IT>V<IT>×</IT>[Urea]<SUB>a</SUB> 
where V is the volume of urea distribution in the whole body and is assumed to be 600 ml/kg (19).

Data analysis. Experiments consisted of four sampling periods. These were the 30-min basal period and the 25- to 50-min, 75- to 100-min, and 125- to 150-min exercise periods. Individual samples within a sampling period were pooled and meaned. The values from each period are presented as the change from basal. Statistics were performed using SuperAnova (Abacus Concepts, Berkeley, CA) on a MacIntosh PowerPC. Statistical comparisons between groups and over time were made using ANOVA designed to account for repeated measures. Significant differences were assessed using contrasts solved by univariate repeated measures. Differences were considered significant when P values were <0.05. Data are expressed as means ± SE.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Arterial glucagon and insulin concentrations. Plasma glucagon concentrations rose by twofold over the course of the exercise period in SimG, whereas concentrations were unchanged from basal in BasG (Fig. 1). Concentrations in BasG were significantly less than concentrations in SimG from 75 to 150 min (P < 0.01-0.001). Plasma insulin levels fell similarly in SimG and BasG.


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Fig. 1.   Effect of exercise with somatostatin plus simulated intraportal insulin and either simulated () or basal (open circle ) intraportal glucagon on arterial plasma glucagon (top) and insulin (bottom) concentrations. Data are means ± SE; n = 5 in each group. * Significantly different from basal (P < 0.05-0.001); dagger  significantly different from corresponding interval in simulated glucagon protocol (P < 0.01-0.001).

Arterial glucose concentrations and glucose infusion rates. Basal arterial glucose concentrations were 109 ± 6 and 109 ± 6 mg/dl in SimG and BasG, respectively. Glucose concentrations were stable in both groups throughout the exercise period. No exogenous glucose was required to clamp glucose in SimG, whereas a maximum of 3.1 ± 1.4 mg · kg-1 · min-1 was infused by the end of exercise in BasG.

Portal vein and hepatic artery blood flows. Portal vein (basal values of 20 ± 1 and 18 ± 2 ml · kg-1 · min-1 in SimG and BasG) and hepatic artery blood flows (basal values of 5 ± 1 and 6 ± 1 ml · kg-1 · min-1 in SimG and BasG in the basal state) were unaffected by exercise and were not significantly different between groups.

Arterial Gln concentrations. Arterial Gln fell gradually when the glucagon response was simulated (P < 0.005 at 75-100 min and 125-150 min) but was unchanged from basal when the glucagon response was maintained at basal (Fig. 2). Gln concentrations were significantly higher in BasG than in SimG during the last two exercise intervals (P < 0.001).


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Fig. 2.   Effect of exercise with somatostatin plus simulated intraportal insulin and either simulated (hatched bars) or basal (open bars) intraportal glucagon on arterial blood Gln concentrations. Data are means ± SE; n = 5 in each group. * Significantly different from basal (P < 0.005); dagger  significantly different from corresponding interval in simulated glucagon protocol (P < 0.001).

Liver glutamine kinetics. Basal net hepatic Gln balance (Fig. 3) was -0.5 ± 0.5 and -0.4 ± 0.2 µmol · kg-1 · min-1 in SimG and BasG, respectively, where a negative value reflects net Gln output. With the onset of exercise, net hepatic Gln output changed to net uptake in both groups, a state that remained for the duration of the exercise period. Net hepatic Gln balance was significantly different from basal rates during the entire exercise period in both groups (P < 0.05-0.001). The nature of the change in net hepatic Gln balance was different in SimG and BasG. In BasG, the peak rate of net hepatic Gln uptake was obtained within the first sampling interval. In SimG, the response continued to rise so that the peak values were reached during the last two sampling intervals. This created responses that were significantly different between SimG and BasG at the last two sampling intervals (P < 0.05-0.02).


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Fig. 3.   Effect of exercise with somatostatin plus simulated intraportal insulin and either simulated (hatched bars) or basal (open bars) intraportal glucagon on net hepatic Gln balance (top), unidirectional hepatic Gln uptake (middle), and unidirectional hepatic Gln output (bottom). The latter two variables were calculated using hepatic extraction of stable and naturally occurring isotopes. Data are means ± SE; n = 5 in each group. * Significantly different from basal (P < 0.05-0.001); dagger  significantly different from corresponding interval in simulated glucagon protocol (P < 0.05-0.02).

Unidirectional hepatic Gln uptake (Fig. 3) rose by sixfold over basal in SimG by the last exercise interval. Significant increases were observed at 75-100 min and 125-150 min (P < 0.02-0.001). No significant changes from basal hepatic Gln uptake were present in BasG. Responses differed between SimG and BasG during 75-100 min and 125-150 min (P < 0.05-0.02). Unidirectional hepatic Gln output (Fig. 3) was unaffected by exercise in SimG until the last exercise sampling interval, during which it was increased compared with basal (P < 0.05). Hepatic Gln output was unaffected by exercise in BasG. Hepatic Gln output responses to exercise were not significantly different between groups (P = 0.062 at 125-150 min).

The exercise-induced increment in glucagon resulted in a gradual increase in hepatic fractional Gln extraction (Fig. 4) that reached sevenfold basal by the end of the exercise period. The increment in hepatic fractional Gln extraction was significantly elevated throughout the exercise period in SimG (P < 0.05-0.001). In contrast, this variable was unaffected by exercise when the rise in glucagon was absent. Responses to exercise were significantly different between SimG and BasG at the 75- to 100-min and 125- to 150-min intervals.


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Fig. 4.   Effect of exercise with somatostatin plus simulated intraportal insulin and either simulated (hatched bars) or basal (open bars) intraportal glucagon on unidirectional hepatic fractional Gln extraction measured isotopically. Data are means ± SE; n = 5 in each group. * Significantly different from basal (P < 0.05-0.001); dagger  significantly different from corresponding interval in simulated glucagon protocol (P < 0.05).

Gut Gln kinetics. Exercise increased net gut Gln balance (Table 1) so that an increase in net uptake was present in both SimG (P < 0.05-0.01 throughout the exercise period) and BasG (P < 0.05 at 25- to 50-min and 125- to 150-min intervals). In light of this, it was surprising that there were no significant changes in either unidirectional gut Gln output or uptake in either group (Table 1). Gut fractional Gln extraction was significantly increased with exercise, regardless of the glucagon replacement protocol (Table 1). Significant increases were present in both groups at the last two sampling intervals (P < 0.05-0.01). There were no significant differences between protocols in any variable related to gut Gln kinetics.

                              
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Table 1.   Effect of exercise on gut Gln net balance, output, uptake, and fractional extraction in dogs receiving somatostatin plus exercise-simulated insulin and glucagon and in dogs receiving somatostatin plus exercise-simulated insulin and basal glucagon

Arterial Glu concentrations and net hepatic and gut Glu balances. Arterial blood Glu concentration was unchanged from basal (66 ± 4 µM) in SimG until the last exercise sampling interval (77 ± 8 µM), at which time it was significantly elevated (P < 0.05). Arterial Glu concentration increased throughout the exercise period in BasG (P < 0.05-0.001) from a basal value of 79 ± 8 to 100 ± 8 µM by the end of exercise. The gut was a net producer of Glu at rest (-0.28 ± 0.04 and -0.24 ± 0.06 µmol · kg-1 · min-1 in SimG and BasG), and rates of net gut Glu balance were not significantly affected by exercise or the glucagon replacement protocol. Net hepatic Glu balance indicated a low rate of net uptake at rest in both groups, which was not significantly different from zero. Rates were not significantly changed with exercise in SimG but became net output at exercise sampling intervals of 25-50 min (-0.08 ± 0.04 µmol · kg-1 · min-1) and 75-100 min (-0.16 ± 0.06 µmol · kg-1 · min-1) in BasG (P < 0.05). BasG was significantly different from SimG at the 75- to 100-min sampling interval (P < 0.05).

Arterial urea concentrations and net hepatic urea output. Arterial urea concentration (Fig. 5) was unaffected by exercise in SimG until the last sampling interval, at which time urea was increased (P < 0.001). Arterial urea concentration was constant throughout the exercise period in BasG. There were no differences in arterial urea concentration between groups. Net hepatic urea output (Fig. 6) rose in response to exercise in SimG by ~10 µmol · kg-1 · min-1 (P < 0.01-0.05 at all exercise sampling intervals) but was unchanged by exercise in BasG. A significant difference between groups was present at the 25- to 50-min interval (P < 0.05). The formation of urea from the Gln amide nitrogen occurred at a fourfold greater rate in SimG than in BasG (Fig. 7).


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Fig. 5.   Effect of exercise with somatostatin plus simulated intraportal insulin and either simulated (hatched bars) or basal (open bars) intraportal glucagon on arterial blood urea concentration. Data are means ± SE; n = 5 in each group. * Significantly different from basal (P < 0.001).



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Fig. 6.   Effect of exercise with somatostatin plus simulated intraportal insulin and either simulated (hatched bars) or basal (open bars) intraportal glucagon on net hepatic urea output. Data are means ± SE; n = 5 in each group. * Significantly different from basal (P < 0.05-0.01); dagger  significantly different from corresponding interval in simulated glucagon protocol (P < 0.05).



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Fig. 7.   Effect of exercise with somatostatin plus simulated intraportal insulin and either simulated (hatched bar) or basal (open bar) intraportal glucagon on formation of urea from Gln amide nitrogen over a 150-min exercise period. Data are means ± SE; n = 5 in each group. dagger  Significantly different from corresponding rate in simulated glucagon protocol (P < 0.05).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Arteriovenous and stable isotopic techniques were used to gain an understanding of the role of glucagon in hepatic glutamine metabolism during exercise. Exercise is a unique model for studying glucagon action, because it allows for an increment in glucagon to be studied without creating hyperglycemia or hyperinsulinemia, both of which may influence Gln metabolism (1). The absence of these two factors that suppress hepatic glucagon action allows the full effect of an increment in glucagon to be assessed. Moreover, the dog model permits not only the measurement of hepatic balance but also the replacement of glucagon into its physiological entry site in the portal vein. This enables glucagon replacement to normal portal vein levels without creating peripheral hyperglucagonemia. Experiments presented with the use of these tools clearly illustrate that a physiological increase in glucagon plays a major role in regulation of hepatic Gln uptake and incorporation of its amide nitrogen to urea. Gln is one of two amino acids that are of primary importance for nitrogen delivery from peripheral tissue to liver. We have shown in previous studies that the net hepatic uptake and fractional extraction of the other amino acid, alanine, is predominantly controlled by glucagon during exercise as well (26). The fact that an increase in glucagon stimulates both hepatic alanine and Gln uptake highlights the effects of glucagon stimulation on both system A and system N transporters, respectively (15), and pathways of metabolism (27). Unidirectional hepatic Gln uptake and fractional extraction rose by ~sixfold and ~sevenfold, respectively, during the last 25 min of exercise when the glucagon response to exercise was simulated. These variables were unchanged when glucagon concentrations were maintained at basal throughout the exercise period. The rise in glucagon during exercise also resulted in a small increase in hepatic Gln output at the last exercise time interval. This is consistent with the demonstration that a rise in glucagon stimulates flux through the Gln synthetase reaction in the perfused rat liver at Gln delivery rates similar to those of the present study (13).

The finding that the increase in glucagon controls hepatic Gln uptake is consistent with previous experiments conducted in vitro (3, 13, 21). It has been shown that glucagon leads to an acute stimulation of net Gln uptake by the isolated perfused rat liver, which is related to an increase in phosphate-dependent glutaminase activity (3) and flux through the glutaminase step (13). It was recently shown, moreover, that [15N]urea formation from [2-15N]- and [5-15N]Gln is stimulated by glucagon in the perfused rat liver (3). One result of glucagon-stimulated hepatic Gln uptake and fractional uptake was that circulating Gln fell during exercise, whereas it was constant when glucagon levels were not increased. This is consistent with studies showing that an increment in glucagon decreases arterial Gln levels in the resting state (2, 6, 23) and increases hepatic gluconeogenesis from Gln (23).

By comparing net hepatic urea output (Fig. 6) and rate of urea formation from Gln amide nitrogen (Fig. 7), we can estimate the importance of Gln amide nitrogen to hepatic ureagenesis. In the group in which the glucagon response was simulated, ~25% of the urea nitrogen came from the Gln amide nitrogen. In the basal glucagon group, the contribution was somewhat lower at ~10 to 15%. When we consider the number of amino acids that could potentially play a role in nitrogen delivery, that any one amino acid contributes as much as 25% under physiological conditions clearly makes it an important vehicle in nitrogen transport and metabolism.

It is well known that Gln is a major oxidative fuel for the small intestine (29). This is reflected by the presence of net and unidirectional gut Gln uptake in basal and exercise states. These data also confirm earlier studies showing that exercise increases net (24) and unidirectional (8) gut fractional Gln extraction. In the present study, gut fractional Gln extraction rose by two- to threefold. The mechanism for this increase does not involve the increment in glucagon, because it occurs even when the glucagon response to exercise is abolished. From a functional standpoint, it is unclear as to whether this increase in gut fractional Gln extraction is to provide more energy for the gut during exercise or whether it is to facilitate the removal of circulating nitrogenous compounds. This increase in gut fractional extraction decreases the need to have the liver take up Gln. The increase in net gut fractional Gln extraction has been shown to correspond to an increase in net gut ammonia output (24). Because increased portal vein ammonia stimulates Gln uptake in the liver, it is also possible that increased gut ammonia released from intestinal Gln metabolism could facilitate Gln uptake in the liver (4).

Despite alterations in gut and liver Gln kinetics during exercise, arterial blood Gln kinetics are changed very little or not at all (8). Moreover, even though the increase in glucagon caused marked differences in hepatic Gln kinetics in the present study, there was no effect on arterial Gln turnover. These findings suggest that arterial blood samples are relatively insensitive to acute changes in splanchnic Gln metabolism. The reason for this may relate to an inability of a vena cava isotope infusion to accurately mimic splanchnic Gln production and utilization. This is consistent with studies conducted in human subjects, where changes in Gln turnover have not been detectable at times when changes might be anticipated. In two separate studies, an infusion of glucagon that caused decrements in arterialized blood Gln levels did not significantly change measured rates of Gln appearance or disappearance (2, 23). In one of these studies, changes in Gln appearance and disappearance were not present, even though glucagon stimulated gluconeogenesis from Gln (23). In another instance, no difference in arterial Gln turnover was evident in exercising human subjects (28), even though increased regional Gln kinetics have been reported to occur (7, 8). Nevertheless, the modified one-compartment isotope dilution method has been shown to accurately trace increases in Gln infusion rate by use of Gln labeled with isotopes of nitrogen or carbon (16). One cannot rule out the possibility, therefore, that reciprocal changes occurring in other tissues (e.g., kidney and muscle) are enough to counterbalance the kinetic changes of Gln in the splanchnic bed. These data also emphasize the difficulty of interpreting isotopic Gln data by use of systemic sampling alone.

Exercise leads to an increase in net hepatic urea output in the dog (8, 24). In contrast, stable isotope-determined urea production was unchanged in human subjects during 3 h of light exercise (40% maximum O2 uptake) and 1 h of heavy exercise (70% maximum O2 uptake) (5). This discrepancy may occur because urea isotope dilution methods have been been shown to dramatically underestimate acute changes in urea production assessed using [13C]urea and calculated using a one-compartment model (9). It may be that the urea space is sufficiently large that it is difficult to trace in the whole body. Studies using other indexes of urea production (i.e., changes in urea excretion, blood levels, and sweat loss) have been shown to increase with exercise (11, 22). The results of the present study show that the exercise-induced increase in net hepatic urea output is reliant on the increase in glucagon levels. Moreover, prevention of the increase in glucagon resulted in a rate of hepatic conversion of Gln to urea during exercise that was only 25% of the normal response. Glucagon has been shown to stimulate ureagenesis in perfused rat liver (13). Furthermore, this hormone was demonstrated to increase the incorporation of 15N-labeled amide nitrogen into urea in isolated rat hepatocytes (3) and perfused rat liver (21). The mechanism for the glucagon-stimulated increase in urea formation appears to be related to an increase in glutaminase activity and an accelerated formation of N-acetylglutamate, an activator of the key ureagenic enzyme, carbamoyl-phosphate synthetase (3).

Finally, it is important to note the difficulty in measuring hepatic balances of cold and isotopic substrates. The signal in balance measurements is the arteriovenous difference, which is generally only a fraction of absolute concentrations. The measurements of Gln and urea balances are particularly vulnerable, because hepatic arteriovenous differences are small, whereas circulating concentrations are high. This creates a particularly low signal-to-noise ratio. We have worked to minimize this problem by pooling samples within an exercise interval. Regardless, one must be particularly wary, under these conditions, of the possibility of missing significant differences when they may truly exist (type I statistical error).

In conclusion, these data confirm that Gln is an important vehicle for nitrogen transport to the liver and show that the exercise-induced increase in glucagon is critical for hepatic Gln metabolism. These data specifically show that the exercise-induced increase in glucagon is 1) essential for the increase in hepatic Gln uptake and fractional extraction, 2) required for the full increment in ureagenesis, 3) required for the specific transfer of the Gln amide nitrogen to urea, and 4) unrelated to the increase in gut fractional Gln extraction. These data show, for the first time by use of the physiological perturbation of exercise, that glucagon is a physiological regulator of hepatic Gln metabolism in vivo.


    ACKNOWLEDGEMENTS

We acknowledge the valuable assistance of Dr. Paul Flakoll and Li Zheng for measurements of glutamine enrichment; Eric Allen, Pam Venson, and Wanda Snead for hormone measurements; and Robert Allison for technical assistance during experiments.


    FOOTNOTES

This research was supported by National Institute of Diabetes and Digestive Diseases Grants R01-DK-47344, R01-DK-50277, and Diabetes Research and Training Center Grant 5-P60-DK-20593.

Address for reprint requests and other correspondence: D. H. Wasserman, Light Hall Rm. #702, Dept. of Molecular Physiology and Biophysics, Vanderbilt Univ. School of Medicine, Nashville, TN 37232-0615 (E-mail: david.wasserman{at}mcmail.vanderbilt.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Received 21 July 1999; accepted in final form 25 April 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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