Hepatic glucose disposition during concomitant portal
glucose and amino acid infusions in the dog
Mary Courtney
Moore1,
Paul J.
Flakoll2,
Po-Shiuan
Hsieh1,
Michael J.
Pagliassotti1,
Doss W.
Neal2,
Michael T.
Monohan1,
Carol
Venable1, and
Alan D.
Cherrington1,2
1 Department of Molecular
Physiology and Biophysics and
2 Diabetes Research and
Training Center, Vanderbilt University, Nashville, Tennessee
37232-0615
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ABSTRACT |
The effect of concomitant intraportal infusion
of glucose and gluconeogenic amino acids (AA) on net hepatic glucose
uptake (NHGU) and glycogen synthesis was examined in 42-h-fasted dogs. After a basal period, there was a 240-min experimental period during
which somatostatin was infused continuously into a peripheral vein and
insulin and glucagon (at 3-fold basal and basal rates, respectively)
and glucose (18.3 µmol · kg
1 · min
1)
were infused intraportally. One group (PoAA,
n = 7) received an AA mixture
intraportally at 7.6 µmol · kg
1 · min
1,
whereas the other group (NoAA, n = 6)
did not receive AA. Arterial blood glucose concentrations and hepatic
glucose loads were the same in the two groups. NHGU averaged 4.8 ± 2.0 (PoAA) and 9.4 ± 2.0 (NoAA)
µmol · kg
1 · min
1
(P < 0.05), and tracer-determined
hepatic glucose uptake was 4.6 ± 1.6 (PoAA) and 10.0 ± 1.7 (NoAA)
µmol · kg
1 · min
1
(P < 0.05). AA data for PoAA and
NoAA, respectively, were as follows: arterial blood concentrations,
1,578 ± 133 vs. 1,147 ± 86 µM
(P < 0.01); hepatic loads, 56 ± 3 vs. 32 ± 4 µmol · kg
1 · min
1
(P < 0.01); and net hepatic uptakes,
14.1 ± 1.4 vs. 5.6 ± 0.4 µmol · kg
1 · min
1
(P < 0.01). The rate of net hepatic
glycogen synthesis was 7.5 ± 1.9 (PoAA) vs. 10.7 ± 2.3 (NoAA)
µmol · kg
1 · min
1
(P = 0.1). In a net sense, intraportal
gluconeogenic amino acid delivery directed glucose carbon away from the
liver. Despite this, net hepatic carbon uptake was equivalent in the
presence and absence of amino acid infusion.
liver; hyperglycemia; liver nerves; glycogen; mixed meal
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INTRODUCTION |
NET HEPATIC GLUCOSE UPTAKE (NHGU) in the conscious dog
is two- to threefold greater in response to intraportal glucose
infusion than in response to peripheral glucose infusion, even when the hormone and glucose levels are made identical (3, 26). The enhancement
of NHGU in response to intraportal glucose delivery is accompanied by
an enhancement of hepatic glycogen synthesis (26).
We have postulated that a signal (termed portal signal) generated
during portal glucose infusion (or absorption of enterally administered
glucose) results in enhanced NHGU (3). Neurophysiological evidence is
consistent with this postulate. For example, the afferent firing rate
in the hepatic branch of the vagus nerve of the guinea pig is inversely
proportional to the portal vein glucose concentration (23). Also,
injection of glucose into the portal vein increases the efferent firing
rate in the vagal pancreatic nerve, whereas injection of the same
amount of glucose into the jugular vein does not (23). Moreover,
functional connections have been identified between glucose-sensitive
afferent neurons in the portal vein and glucose-sensitive neurons in
the central nervous system (2).
During the postprandial period after intragastric administration of a
mixed meal containing protein, glucose, and lipid, NHGU in conscious
dogs was ~80% less than would have been predicted based on the
increases in the insulin concentration (>9-fold basal) and the
hepatic glucose load (~2-fold basal) (20). The rise in glucagon
(~20 pg/ml) during the postprandial period may have contributed to
the unexpectedly low NHGU (20). During intraportal glucose (hepatic
glucose load 2-fold basal) and insulin (4-fold basal) infusion, a 20 pg/ml increase in glucagon reduced NHGU ~50% compared with that
observed in the presence of basal glucagon (16). It is therefore
unlikely that the increase in glucagon which occurred in response to
the mixed meal was sufficient by itself to account for the marked
blunting of NHGU. Another possible explanation for the very small NHGU
observed after consumption of a mixed meal is the potential interaction
between macronutrients when they are delivered simultaneously.
There exists a potential for complex interactions, and possibly
competition, between signals created in response to intraportal delivery of glucose and other substrates (in particular amino acids)
contained in mixed meal feedings. It has been shown, for example, that
intraportal or intraperitoneal injection of solutions of single amino
acids or mixtures of amino acids alters the afferent discharge rate of
the hepatic branch of the vagus nerve (24, 25). It is therefore
possible that net hepatic uptake of substrates may differ when they are
administered singly and when they are administered in combination. We
hypothesized that intraportal delivery of glycogenic substrates other
than glucose (e.g., gluconeogenic amino acids) might suppress NHGU
during intraportal glucose delivery. The aim of the present study was
to quantify NHGU and hepatic glycogen synthesis during concomitant
intraportal delivery of glucose and a mixture of gluconeogenic amino
acids under conditions in which the insulin and glucagon levels were
fixed (3-fold basal and basal, respectively) using somatostatin and
intraportal hormone replacement.
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MATERIALS AND METHODS |
Animals, diets, and experimental preparation.
Studies were carried out on conscious 42-h-fasted adult dogs of either
sex with a mean weight of 22 ± 1 kg. Housing and diet have
previously been described (20). The protocol was approved by the
Vanderbilt University Medical Center Animal Care Committee, and animals
were housed according to American Association for the Accreditation of
Laboratory Animal Care International guidelines. Approximately 16 days
before study, all dogs underwent a laparotomy under general anesthesia,
and silicone rubber catheters (Dow Corning, Midland, MI) were inserted
in the portal and left common hepatic veins, a splenic and a jejunal
vein, and the femoral artery (26). Ultrasonic flow probes (Transonic
Systems, Ithaca, NY) were positioned around the portal vein and hepatic
artery, and their proximal ends were placed in subcutaneous pockets.
Approximately 2 days before study, blood was drawn from each animal. A
dog was studied only if it met established criteria: leukocyte count
<18,000/mm3, hematocrit >35%,
consumption of all of the daily food ration, and normal stools. On the
morning of the study, the proximal ends of the flow probes and
surgically implanted catheters were exteriorized, the catheters were
cleared, the dog was placed in a Pavlov harness, and intravenous access
was established in three peripheral veins.
Experimental design.
At
120 min, a primed (40 µCi), continuous (0.4 µCi/min)
peripheral infusion of
D-[3-3H]glucose
and a continuous peripheral infusion of indocyanine green (ICG; 4 µg · kg
1 · min
1;
Becton Dickinson, Cockeysville, MD) dye were begun. The latter provided
confirmation of hepatic vein catheter placement and a second
measurement of hepatic blood flow. After 80 min (
120 to
40) of dye equilibration, there was a 40-min (
40 to 0)
control or basal period followed by a 240-min (0 to 240) experimental period. At time 0, constant infusions
of several solutions were begun, and these infusions continued for the
next 240 min (experimental period). Somatostatin (0.8 µg · kg
1 · min
1;
Bachem, Torrance, CA) was infused to suppress endogenous insulin and
glucagon secretion. Insulin (7.2 pmol · kg
1 · min
1;
3-fold basal) and glucagon (both hormones obtained from Eli Lilly,
Indianapolis, IN) were delivered into the portal circulation via the
jejunal and splenic infusion catheters. In two dogs in each group, the
rate of glucagon infusion was 0.65 ng · kg
1 · min
1.
This rate resulted in circulating glucagon levels slightly higher than
basal, however, and therefore the infusion rate was lowered to 0.5 ng · kg
1 · min
1
in the remainder of the dogs. Dextrose (20%, 18.3 µmol · kg
1 · min
1;
Baxter Healthcare, Deerfield, IL) mixed with
p-aminohippuric acid (PAH; delivered
at 1.7 µmol · kg
1 · min
1
Sigma, St. Louis, MO) was also infused intraportally. PAH was used to
assess mixing of the infused glucose with blood in the portal and
hepatic veins as described previously (26). In one group of dogs (PoAA,
n = 7), a mixture of gluconeogenic
amino acids (L-isomers of
glutamine, glutamate, threonine, serine, glycine, and alanine; molar
ratio 1.0, 0.2, 0.5, 0.2, 0.4, and 0.4, respectively) was infused
intraportally at 7.6 µmol · kg
1 · min
1.
The ratios and rate were chosen to mimic the ratios and the absorption
rates that we observed previously after delivery of a liquid mixed meal
to conscious dogs (20). The amino acid mixture was prepared just before
time 0 by dissolving the individual
crystalline amino acids (Sigma) in deionized water. The second group
(NoAA, n = 6) received intraportal
saline rather than amino acids. In addition to the constant infusions,
a primed, continuous peripheral infusion of 50% dextrose was begun in
both groups at time 0, so that the
blood glucose could be quickly clamped at its desired value. Blood
samples of 0.2 ml were obtained from the artery every 5 min to permit
measurement of the plasma glucose concentration, and the peripheral
glucose infusion rate was adjusted on the basis of these measurements
to maintain the hepatic glucose load at 1.5-fold basal throughout the
experimental period. Larger blood samples (4-9 ml) for data
acquisition were obtained from the artery, portal vein, and hepatic
vein every 20 min during the basal period and every 15-30 min
during the experimental period. The collection, processing, and
analysis of blood samples have been described in detail elsewhere (20).
After completion of the experiment, each animal was killed with an
overdose of pentobarbital, the liver was removed, and a tissue sample
from each liver lobe was freeze clamped within 5 min of pentobarbital
administration and stored at
70°C to await analysis.
Processing and analysis of samples.
Blood glucose, lactate, alanine, glycerol, and hematocrit; plasma
glucose, insulin, and glucagon concentrations; plasma
[3H]glucose specific
activity; and liver glycogen concentrations were determined as
described previously (20). HPLC was used to determine amino acid
concentrations on sulfosalicylic acid-deproteinized blood samples (32).
Blood glutamine and glutamate concentrations were measured on
perchloric acid-deproteinized samples (20). PAH was measured in
perchloric acid-deproteinized blood as previously described (26).
Calculations.
The thoroughness of mixing of the infused glucose in the portal vein
was assessed by comparing recovery of PAH (which was mixed with
glucose) in the portal and hepatic veins with the PAH infusion rate
(26). Because of the magnitude of the coefficient of variation of the
method for assessing PAH balance, samples were considered statistically
unmixed (>95% confidence that mixing did not occur) if hepatic or
portal vein recovery of PAH was 40% greater or less than the actual
amount of PAH infused. An experiment was defined as having poor mixing
(and was excluded from data base) if poor mixing was observed at at
least three of the eight time points in the experimental period. In
PoAA mixing of infused amino acids was considered to have occurred if
mixing of glucose occurred in the same animal, since glucose and amino
acids were infused through the same catheters. Seventeen dogs were
studied; one is not included in the data base because of malfunction of sampling catheters, and three were excluded because of poor mixing. In
the 13 animals included in the data base, mixing failed to occur at
<10% of the time points. Because mixing errors, when they occurred,
were random, individual data points were not excluded if the experiment
as a whole was included. Good mixing was evident in both groups. The
ratio of recovered to infused PAH in the portal and hepatic veins was
0.9 ± 0.1 and 0.9 ± 0.1, respectively (with a ratio of 1.0 representing ideal mixing).
Hepatic blood flow (HBF) was calculated by two methods, ultrasonic flow
probes and dye extraction (20). The results obtained with ultrasonic
flow probes and dye were not significantly different. Because the flow
probes make it possible to determine the relative proportions of the
hepatic blood flow provided by the hepatic artery and the portal vein,
calculations reported in here utilize HBF obtained from the flow probes
when flow probes were available. One or the other of the flow probes
did not function in two dogs in PoAA and three in NoAA. In these
animals, ICG-derived flows were used, and the portal vein was assumed
to provide 80% of hepatic blood flow during the basal period and 74%
during the experimental period. These assumptions were based on the
findings in the present studies in the animals having functional flow
probes and were also consistent with our findings in previous studies
conducted in the presence and absence of somatostatin (e.g., Refs. 21, 26).
The rate of substrate delivery to the liver, or hepatic substrate load,
was calculated by a direct (d) method as
where
[S] is the substrate concentration, A and P refer to artery
and portal vein, respectively, and ABF and PBF refer to blood flow
through the hepatic artery and portal vein, respectively. To avoid any
potential errors arising from either incomplete mixing of glucose
during intraportal infusion or lack of precise measurements of the
distribution of hepatic blood flow, hepatic glucose load was also
calculated by an indirect (i) method
where
G is the blood glucose concentration,
GIRPo is the intraportal glucose
infusion rate, and GUG is the uptake of glucose by the gastrointestinal
tract, calculated based on the previously described relationship
between the arterial blood glucose concentration and GUG (26).
The load of a substrate exiting the liver was calculated as
where
H represents the hepatic vein.
Direct and indirect methods were used in calculation of net hepatic
balance (NHB). The direct calculation was as follows: NHBd = loadout
loadin(d). The indirect
calculation was as follows: NHBi = loadout
loadin(i). A negative value
indicates net uptake. Both equations were used in calculation of net
hepatic glucose and amino acid balance, but only the direct calculation
was employed for other substrates. The results for NHB of glucose and
amino acids did not differ regardless of the method used in
calculation. The results given in this report utilize the indirect
calculation, because this method is less likely to be affected by any
inadequate mixing of infused substrates in the portal vein.
Net fractional substrate extraction by the liver was calculated
directly and indirectly as the ratio of NHB to
loadin.
Endogenous rate of appearance (Endo
Ra) was calculated using a
two-compartment model (18) with canine parameters (10), deducting the
rate of exogenous (portal and peripheral) glucose infusion. The rate of
hepatic glucose uptake was calculated as the balance of
D-[3-3H]glucose
across the liver, using the same formula as for NHB but substituting
hepatic plasma flow measurements. The results were divided by the
weighted inflowing glucose specific activity (expressed as dpm/µmol
glucose). The weighted specific activity was the sum of the arterial
and portal glucose specific activities, weighted for the proportion of
flow provided by each vessel. These calculations assume that
3H measurements were obtained
before dilution of the tracer glucose resulting from the addition of
unlabeled glucose to the plasma as it passes through the liver.
Nevertheless, the calculation should be accurate even if the assumption
is not correct, since there was little or no change in glucose specific
activity across the liver.
The trapezoidal rule was used to determine the area under the curve
(AUC). Both positive and negative excursions (i.e., net balance) were
included in the calculations for net hepatic disposition of substrates.
Net hepatic glycogen synthesis was calculated as the difference between
poststudy glycogen concentrations in the glucose-infused dogs and basal
concentrations in 11 dogs killed after a 42-h fast (corresponding to
time 0 in experimental animals) (21).
The glycogen concentrations for each dog represent the mean of the values for the seven liver lobes, weighted for the percentage of liver
mass accounted for by each lobe (21). The contribution of the direct
pathway of glycogen synthesis (glucose
glucose
6-phosphate
glucose 1-phosphate
UDP-glucose
glycogen) was also assessed by
dividing the number of 3H dpm in
the liver by the average inflowing
[3-3H]glucose specific
activity.
Data are presented as means ± SE. SYSTAT (Evanston, IL) was used
for statistical analysis. Time-course data were analyzed with
repeated-measures ANOVA with post hoc analysis by univariate F tests. Independent-sample
t-tests were used for analysis of glycogen data and comparison of AUC. Results were considered
statistically significant at P < 0.05.
 |
RESULTS |
Plasma insulin and glucagon concentrations.
Plasma insulin concentrations in the two groups did not differ at any
time (Fig. 1). Arterial plasma insulin
concentrations during the experimental period (158 ± 12 pM in PoAA
and 146 ± 17 pM in NoAA) were ~3- to 3.5-fold basal, mimicking
concentrations in the postprandial state. Arterial plasma glucagon
levels remained at basal throughout the study in both groups and did
not differ between groups at any time.

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Fig. 1.
Arterial plasma insulin and glucagon concentrations in 42-h-fasted dogs
during basal sampling period ( 40 to 0 min) and experimental
period (0-240 min). During experimental period, all dogs received
a peripheral infusion of somatostatin and intraportal infusions of
insulin (4-fold basal), glucagon (basal), and glucose (18.3 µmol · kg 1 · min 1).
PoAA group (n = 7) received an
intraportal infusion of gluconeogenic amino acids (7.6 µmol · kg 1 · min 1);
NoAA group (n = 6) received no amino
acid infusion. There are no significant differences between groups.
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Hepatic blood flow, blood glucose metabolism, and glucose infusion
rates.
Total HBF did not differ among the groups at any time (mean: 28 ± 2 and 25 ± 3 ml · kg
1 · min
1
in PoAA and NoAA, respectively), nor did it change significantly between the basal and experimental periods in either group (data not
shown).
Arterial blood glucose concentrations in the groups were similar
throughout the studies (basal concentrations: 4.2 ± 0.1 and 4.2 ± 0.2 mM in PoAA and NoAA, respectively; experimental period concentrations: 6.2 ± 0.2 and 6.5 ± 0.4 mM in PoAA and NoAA,
respectively; Fig. 2). The mean hepatic
glucose loads were 130 ± 12 (PoAA) and 133 ± 18 (NoAA)
µmol · kg
1 · min
1
during the basal period and 205 ± 18 (PoAA) and 203 ± 29 (NoAA) µmol · kg
1 · min
1
during the experimental period (NS; Fig. 2). The peripheral glucose infusion rates required to maintain this increase in the hepatic glucose load were ~40% less in PoAA (Table
1), but the variance among animals resulted
in a P value of 0.3.

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Fig. 2.
Arterial blood glucose concentrations and hepatic glucose load. See
legend to Fig. 1 for description of study design. There are no
differences between groups.
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Table 1.
Peripheral glucose infusion rates in dogs receiving intraportal glucose
infusion with or without concomitant intraportal infusion of
gluconeogenic amino acids
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Net hepatic glucose output was similar in the two groups during the
basal period (9.8 ± 2.0 and 10.1 ± 1.7 µmol · kg
1 · min
1
in PoAA and NoAA, respectively; Fig. 3).
During the experimental period, NHGU
(µmol · kg
1 · min
1) averaged 4.8 ± 2.0 in PoAA and 9.4 ± 2.0 in NoAA
(P < 0.05). (As previously stated in
MATERIALS AND METHODS, these results were obtained using the indirect calculation. The direct calculation yielded a difference of 5.5 ± 2.1 µmol · kg
1 · min
1
in NHGU between the groups during the experimental period, which was
not different from the results with the indirect calculation.) The
net hepatic fractional extraction of glucose (Table
2) during the experimental period in PoAA
was approximately one-half of that in NoAA (2.6 ± 0.7% vs. 4.8 ± 0.9%; P < 0.05).

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Fig. 3.
Net hepatic balance of glucose and gluconeogenic amino acids. See
legend to Fig. 1 for description of study design. Net hepatic glucose
uptake was significantly greater (P < 0.05) in NoAA, and net hepatic uptake of gluconeogenic amino acids
was greater (P < 0.01) in PoAA.
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Table 2.
Net hepatic fractional extraction of glucose in dogs receiving
intraportal glucose infusion with or without a concomitant
intraportal infusion of gluconeogenic amino acids
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Endo Ra decreased similarly in
both groups during the experimental period to rates that were no
different from zero (Table 3). The mean
tracer-determined hepatic glucose uptake rates during the experimental
period were 4.8 ± 1.4 and 10.4 ± 2.0 µmol · kg
1 · min
1
in PoAA and NoAA, respectively (Table 3).
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Table 3.
Net hepatic glucose balance, endogenous Ra, and
tracer-determined hepatic glucose uptake in dogs receiving a
gluconeogenic amino acid mixture or no amino acids
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Gluconeogenic amino acid metabolism.
During the basal period, parameters related to gluconeogenic amino acid
metabolism (arterial and portal blood concentrations, hepatic load, net
hepatic uptake, and net hepatic fractional extraction; Table
4, Figs. 3 and
4) were similar in the two groups. During the experimental period, however, arterial and portal blood amino acids
concentrations were higher in PoAA than NoAA (mean values for total of
gluconeogenic amino acids in artery: 1,573 ± 133 vs. 1,147 ± 86 µM, P < 0.05; in portal vein:
2,055 ± 175 vs. 1,137 ± 85, P < 0.05). Similarly, the hepatic load of gluconeogenic amino acids
(Fig. 4) was higher (P < 0.01) in
PoAA at every time point during the experimental period (mean values
were 56 ± 3 and 32 ± 4 µmol · kg
1 · min
1
in PoAA and NoAA, respectively). As would be expected with the greater
hepatic load of amino acids in PoAA, the rate of net hepatic amino acid
uptake was greater in PoAA than in NoAA throughout the experimental
period (P < 0.01; mean rates for
total gluconeogenic amino acid uptake were 14 ± 2 and 6 ± 1 µmol · kg
1 · min
1
in PoAA and NoAA, respectively; Fig. 3). In both groups, the net
hepatic fractional extraction of alanine, glutamate, glutamine, and
serine increased significantly (P < 0.05) compared with baseline, and there was a trend toward an increase
in net hepatic fractional extraction of glycine (Table 4). The
experimental period values for net hepatic fractional extraction of
threonine were similar in the two groups, but the change from baseline
reached statistical significance only in PoAA.
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Table 4.
Arterial and portal blood concentrations, net hepatic uptake, and net
hepatic fractional extraction of individual gluconeogenic amino
acids in dogs receiving intraportal glucose infusion with or without a
concomitant intraportal infusion of gluconeogenic amino acids
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Fig. 4.
Arterial and portal vein concentrations and hepatic load of
gluconeogenic amino acids (serine, threonine, glutamine, glutamate,
glycine, and alanine). See legend to Fig. 1 for description of study
design. Values were significantly greater in PoAA than NoAA during
experimental period (arterial and portal concentrations,
P < 0.05; hepatic load,
P < 0.01).
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Lactate metabolism.
There were no differences between groups in arterial blood lactate
concentrations or net hepatic lactate balance. Both groups exhibited
net hepatic lactate uptake (~7
µmol · kg
1 · min
1)
during the basal period, shifted to net hepatic lactate output within
30 min of the start of the experimental period, and returned to net
hepatic lactate uptake before the end of the study (data not shown).
Net hepatic glycogen synthesis.
The mean rates of net hepatic glycogen synthesis were 7.5 ± 1.9 and
10.7 ± 2.3 µmol glucosyl
residues · kg
1 · min
1
in PoAA and NoAA, respectively (P = 0.1). The hepatic 3H content
(expressed per kg body wt) and the inflowing plasma [3H]glucose specific
activities were 939,076 ± 220,971 dpm and 1,165 ± 220 dpm/µmol in PoAA and 992,130 ± 407,759 dpm and 612 ± 233 dpm/µmol in NoAA. Glycogen synthesis via the direct pathway, measured by deposition of tritiated glycogen, averaged 3.4 ± 0.4 and 6.8 ± 0.5 µmol glucosyl
residues · kg
1 · min
1
in PoAA and NoAA, respectively (P < 0.01).
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DISCUSSION |
NHGU was reduced 50% in the group receiving intraportal amino acid
infusion (4.8 ± 2.0 vs. 9.4 ± 2.0 µmol · kg
1 · min
1,
P < 0.05), and tracer-determined
hepatic glucose uptake was reduced by a similar amount. Normally
glucagon increases in the postprandial period after intake of glucose
and amino acids or a mixed meal (20) as a result of the stimulatory
effect of amino acids on glucagon secretion. If we had increased
circulating glucagon levels, rather than maintaining basal
glucagonemia, it could be anticipated that NHGU would have been reduced
even further (16), thus increasing the impact of amino acids on glucose
handling by the liver.
The amino acid infusion maintained relatively stable circulating levels
of gluconeogenic amino acids in PoAA, but in NoAA the amino acid
concentrations declined significantly in response to hyperinsulinemia,
hyperglycemia, and the portal signal. The fall in amino acid
concentrations was associated with an increase in their net hepatic
fractional extraction (Table 4). Levels of gluconeogenic amino acids
have been observed to decline ~20%
50% from basal values in
the presence of physiological hyperinsulinemia (peripheral insulin
concentrations of 120-240 pM) in the rat, dog, and human (12, 17,
19, 28). Although insulin is an upregulator of hepatic system A
activity, it does not affect other amino acid transport systems such as
ASC, N, or L (11). Thus, other than a small effect on alanine, insulin
has little capacity to alter net hepatic fractional extraction of amino
acids. In the presence of a euglycemic, hyperinsulinemic (~120 pM)
clamp, the fractional extraction of gluconeogenic amino acids did not change compared with basal conditions (28). Therefore the increase in
fractional extraction of the amino acids in the current report is
unlikely to be due merely to hyperinsulinemia. On the other hand,
hyperglycemia by itself is also unable to alter the fractional extraction of amino acids. During a hyperglycemic (~10.5 mM), euinsulinemic clamp in conscious dogs, the fractional extraction of the
gluconeogenic amino acids remained stable (29). It therefore appears
that the combination of hyperinsulinemia and hyperglycemia or the
presence of the portal signal must have been responsible for the
enhanced net fractional extraction of the amino acids by the liver.
There are several possible reasons for the inhibition of NHGU in the
PoAA group, including substrate competition, a relative insulin
resistance, and neural signals induced by amino acid infusion (14, 25).
The possibility of substrate competition cannot be adequately assessed
from the current studies. If the glycogen content is indeed lower in
PoAA, then a mechanism other than or in addition to must have been
operating. As to the possibility of insulin resistance, investigators
using the hyperinsulinemic-euglycemic clamp technique in the human have
reported that amino acids caused insulin resistance both by reducing
whole body glucose utilization and by interfering with suppression of
hepatic glucose production by insulin, primarily by enhancing
gluconeogenesis (1, 31). However, glucagon was not controlled, and
glucagon levels were consistently higher during amino acid delivery
than in control studies without amino acids (1, 31). A selective
increase in glucagon in subjects receiving an amino acid load was found to enhance endogenous glucose production and inhibit amino acid-induced protein synthesis (8). When glucagon was maintained at basal concentrations in human subjects receiving infusions of somatostatin, 10-fold basal insulin, a balanced amino acid mixture, and glucose to
maintain euglycemia, there was no reduction in glucose disposal, and
endogenous glucose production was fully suppressed (6). Moreover, use
of tracer-determined glucose oxidation (to overcome inherent
limitations of indirect calorimetry in measuring substrate oxidation
rates during amino acid administration) demonstrated no reduction of
glucose oxidation during amino acid infusion in humans (30). In the
current study, the peripheral glucose infusion rate required to
maintain the hepatic glucose load at 150% basal did not differ between
groups, although it tended to be lower in the group receiving amino
acids. Thus we have no clear evidence that amino acid infusion resulted
in insulin resistance.
Finally, amino acids might have inhibited NHGU by eliciting a neural
signal that conflicted with or modulated the "portal signal"
created by intraportal glucose infusion. Neural sensors for many amino
acids have been identified in the portal region (24, 25, 27).
Intraportal injection of several amino acids, including glycine and
threonine, results in dose-dependent depression of the afferent firing
rate in the hepatic branch of the vagus nerve (25). In contrast,
intraportal injection of other amino acids, including alanine and
serine, increases the afferent hepatic branch firing rate (25). The
efferent firing rates in the pancreatic branches of the vagal and
splanchnic nerves change inversely and directly, respectively, with the
afferent firing rate in the hepatic vagal branch, suggesting that
sensors in the hepatoportal system reflexively regulate the
neuroendocrine response to amino acid-containing feedings (23). Neural
signals resulting from intraportal substrate delivery also appear to
affect the liver directly. In the absence of hepatic nerves, NHGU
during intraportal glucose delivery in conscious dogs is blunted
compared with the response in the presence of hepatic nerves, even with
insulin and glucagon concentrations fixed at similar levels in the
hepatic-innervated and -denervated groups (4). In addition to the
hepatoportal region, the amino acids could also have been sensed in
other sites. For example, they might have affected the central nervous
system directly. Also, the concentrations of the gluconeogenic amino
acids were higher in the arterial circulation in PoAA than in NoAA, and
thus we cannot rule out a peripheral effect of the amino acids. Further studies are underway in our laboratory to explore this question more
thoroughly. However, functional evidence suggests that the portal
delivery route may have a unique role to play in hepatic amino acid
metabolism. Intraportally delivered leucine stimulated hepatic
fibrinogen synthesis in conscious dogs, whereas peripherally infused
leucine had no such effect (5).
It is not clear whether it was one or two amino acids, or the
combination of the six amino acids, that were responsible for the
results obtained. It is noteworthy that fractional extraction of
glutamine and glutamate increased during intraportal infusion of amino
acids (Table 4). Glutamate is recognized as a neurotransmitter in many
sites (7), and there is evidence that glutamine can modulate both the
intestinal absorption and hepatic uptake of glucose. Delivery of
glutamine into the intestinal lumen, but not into the superior
mesenteric artery, was found to increase hepatic glucose uptake in a
combined liver-intestine perfusion preparation in which both the
hepatic and intestinal nerve plexuses remained intact but not in an
isolated (and denervated) liver perfusion system (13). This suggests
that enterohepatic nerves, humoral factors, or absorbed substrates may
act singly or jointly to regulate hepatic glucose uptake during enteral
administration of glutamine. NHGU was decreased, rather than increased,
during intraportal amino acid infusion in the current studies,
indicating that enteric control mechanisms had been bypassed, a mixture
of amino acids may produce different effects than glutamine given alone, or the effect of glutamine on NHGU may be dosage dependent, as
shown in vitro (13).
Net hepatic glycogen synthesis was reduced ~30% in the animals
receiving the amino acid infusion relative to those not receiving amino
acids, although this reduction did not reach statistical significance
(P = 0.1). This is consistent with our
previous findings that net hepatic glycogen synthesis is strongly
correlated with NHGU in dogs receiving glucose infusion (peripherally
or portally), intraportal insulin (basal or 4-fold basal), and basal
glucagon (26). The failure to achieve statistical significance was
probably a result of a type II statistical error; we calculated the
n required for this study based on the
variance and expected difference in NHGU, the primary variable of
interest. Net hepatic uptake of glycogenic substrates (sum of glucose,
gluconeogenic amino acids, and glycerol) was the same in the two groups
(Fig. 5); thus glycogen accumulation in
PoAA was not impaired by any reduction in net substrate supply to the
liver. In each group, NHGU was sufficient to account for all glycogen
deposition via the direct pathway, as well as net hepatic release of
lactate during the experimental period. Net hepatic glycogen synthesis
and lactate production accounted for 88% of the net hepatic carbon
uptake by the NoAA group. Much of the carbon remaining unaccounted for
(~1.5 µmol glucose
equivalents · kg
1 · min
1)
was undoubtedly oxidized, with a small amount being utilized for
lipogenesis (20). In the PoAA group, only 70% of net hepatic substrate
uptake could be accounted for by net hepatic glycogen synthesis and
lactate release. We have previously observed that the postprandial
hepatic oxidation rate averaged ~1.5 µmol glucose equivalents · kg
1 · min
1
and that very little de novo lipogenesis occurred in the liver in a
group of dogs receiving an intragastric mixed meal in which glucose
provided all the carbohydrate (20). If hepatic oxidation and lipogenic
rates are assumed to be the same in PoAA and mixed-meal-fed dogs,
~2 µmol glucose
equivalents · kg
1 · min
1
were available for purposes other than glycogen formation,
lactate production, and oxidation in the livers of PoAA. There is no
evidence of enhanced gluconeogenesis in the PoAA group; Endo
Ra fell to a similar, low rate in
both groups (Table 3). Administration of amino acids may have
stimulated hepatic protein synthesis and/or reduced hepatic
protein breakdown. Humans receiving a meal containing glucose, lipid,
and amino acids were observed to have significantly enhanced synthesis
of albumin compared with subjects receiving an isocaloric meal
containing only glucose and lipid (9, 33). The combination of amino
acid delivery and hyperinsulinemia enhanced albumin synthesis more than
hyperinsulinemia alone (33). Selective hypothreoninemia or
hypoisoleucinemia for 4 h impaired whole body protein synthesis but not
hepatic protein synthesis (17). Thus, even though the amino acid
mixture administered in these studies was lacking in most of the
essential amino acids, an enhancement of intrahepatic protein synthesis
may have occurred, with intrahepatic proteolysis, or possibly
proteolysis in other tissues, providing the additional amino acids
needed. On the other hand, a specific group of regulatory amino acids,
including glutamine, has been observed to inhibit hepatic protein
degradation (22), and thus a decrease in the rate of autophagy may
explain the greater hepatic amino acid retention in the group receiving
amino acids. The mechanism or mechanisms responsible for determining
the fate of carbon entering the liver remain to be elucidated, but
hepatic nerves present one possibility for directing carbon away from
one biosynthetic pathway (e.g., glycogenosis) and into another (e.g.,
protein synthesis).

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|
Fig. 5.
Summary of net hepatic uptake of glycogenic precursors, glucose,
gluconeogenic amino acids, and glycerol, in dogs receiving intraportal
glucose infusion with (PoAA, n = 7) or
without (NoAA, n = 6)
concomitant intraportal gluconeogenic amino acid infusion. Total net
hepatic glycogenic precursor uptake did not differ between groups.
|
|
In a net sense, intraportal infusion of gluconeogenic amino acids
concurrent with intraportal glucose infusion directs glucose carbon
away from the liver and toward other tissues but causes a reciprocal
increase in gluconeogenic amino acid uptake by the liver. Thus net
hepatic uptake of glycogenic carbon is equivalent in animals receiving
intraportal glucose with or without concomitant intraportal infusion of
amino acids. Despite this, the animals receiving amino acids deposited
~30% less hepatic glycogen (P = 0.1), probably because of increased requirements for carbon to be used
for hepatic protein synthesis or because of a decrease in intrahepatic
proteolysis. These data are consistent with the existence of a
generalized energy receptor (24), or a coordinated system of receptors
for individual substrates, which is responsible for determining the
contribution of the liver to substrate disposition.
 |
ACKNOWLEDGEMENTS |
The authors appreciate the assistance of Jon Hastings, Wanda Snead,
and Pam Venson.
 |
FOOTNOTES |
This work was supported by National Institute of Diabetes and Digestive
and Kidney Diseases Grants R-01-DK-43706 and DK-40936 and Diabetes
Research and Training Center Grant DK-20593.
A preliminary report of this work was presented at the American
Diabetes Association Annual Meeting, Atlanta, GA, June 1995, and
published in abstract form (Diabetes
44, Suppl. 1: 90A, 1995).
Present address of M. J. Pagliassotti: Sect. of Pediatric Nutrition,
Dept. of Pediatrics, University of Colorado Health Sciences Center,
Denver, CO 80262.
Address for reprint requests: M. C. Moore, 702 Light Hall, Dept. of
Molecular Physiology and Biophysics, Vanderbilt University School of
Medicine, Nashville, TN 37232-0615.
Received 14 July 1997; accepted in final form 23 January 1998.
 |
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