Inhibition of glycogenolysis enhances gluconeogenic precursor
uptake by the liver of conscious dogs
Masakazu
Shiota,
Patricia A.
Jackson,
Hilmar
Bischoff,
Michael
McCaleb,
Melanie
Scott,
Michael
Monohan,
Doss W.
Neal, and
Alan D.
Cherrington
Department of Molecular Physiology and Biophysics, Vanderbilt
University School of Medicine, Nashville, Tennessee
37232-0615; and Bayer Research Center, West Haven, Connecticut
06516-4175
 |
ABSTRACT |
We investigated the effect of inhibiting
glycogenolysis on gluconeogenesis in 18-h-fasted conscious dogs with
the use of intragastric administration of BAY R 3401, a glycogen
phosphorylase inhibitor. Isotopic
([3-3H]glucose and
[U-14C]alanine) and
arteriovenous difference methods were used to assess glucose
metabolism. Each study consisted of a 100-min equilibration, a 40-min
control, and two 90-min test periods. Endogenous insulin and glucagon
secretions were inhibited with somatostatin (0.8 µg · kg
1 · min
1),
and the two hormones were replaced intraportally (insulin: 0.25 mU · kg
1 · min
1;
glucagon: 0.6 ng · kg
1 · min
1).
Drug (10 mg/kg) or placebo was given after the control period. Insulin
and glucagon were kept at basal levels in the first test period, after
which glucagon infusion was increased to 2.4 ng · kg
1 · min
1;
BAY R 3401 decreased tracer-determined endogenous glucose production [rate of glucose production
(Ra): 14 ± 1 to 7 ± 1 µmol · kg
1 · min
1]
and net hepatic glucose output (11 ± 1 to 3 ± 2 µmol · kg
1 · min
1)
during test
1. It increased the net hepatic uptake
of gluconeogenic substrates from 9.0 ± 2.0 to 11.6 ± 0.6 µmol · kg
1 · min
1.
Basal glycogenolysis was decreased by drug (9.1 ± 0.7 to 1.5 ± 0.2 µmol glucosyl
U · kg
1 · min
1).
Placebo had no effect on Ra or the
uptake of gluconeogenic precursors by the liver. The rise in glucagon
increased Ra by 22 ± 3 and by
8 ± 2 µmol · kg
1 · min
1
(at 10 min) in placebo and drug, respectively. The rise in glucagon caused little change in the net hepatic uptake
(µmol · kg
1 · min
1)
of gluconeogenic substrates in placebo (8.2 ± 0.6 to 9.0 ± 1.0) but increased it markedly (11.6 ± 0.6 to 15.4 ± 1.0) in drug. Glucagon increased glycogenolysis by 22.1 ± 2.5 and by 7.8 ± 1.6 µmol · kg
1 · min
1
in placebo and drug, respectively. The amount of glycogen (µmol glucosyl U/kg) synthesized from gluconeogenic carbon was
four times higher in drug (48.6 ± 9.7) than in placebo (11.3 ± 1.7). We conclude that BAY R 3401 caused a marked reduction in basal and glucagon-stimulated glycogenolysis. As a result of these changes, there was an increase in the net hepatic uptake of gluconeogenic precursors and in glycogen synthesis.
BAY R 3401; glucagon
 |
INTRODUCTION |
THE LIVER PRODUCES GLUCOSE via glycogen breakdown
and/or gluconeogenesis, and the relative contribution of each
to total glucose production changes with altered nutritional and
metabolic states. Several studies in dogs and humans have shown that
increased delivery of gluconeogenic precursors, such as alanine (11,
39), glycerol (20, 38), or lactate (6, 8, 21), to the liver has no
acute effect on the amount of glucose produced by that organ. Delivery
of lactate (8), glycerol (38), or alanine (11) to postabsorptive dogs
in the presence of fixed basal levels of insulin and glucagon increased
both the hepatic uptake of these precursors and their conversion into
glucose but did not change the total rate of glucose production
appreciably. These data support the concept that when gluconeogenesis
increases in the liver, glycogenolysis decreases. Gluconeogenic
precursors can alter hepatic glycogen metabolism not only by exerting
regulatory effects on glycogen phosphorylase and synthase but also by
serving as substrates for glycogen synthesis (for review, see Ref. 40).
The above data suggest the existence of an autoregulatory mechanism
within the liver such that the desired rate of hepatic glucose output can be maintained regardless of the gluconeogenic precursor supply.
Glucagon is a primary determinant of hepatic glucose production (for
review, see Refs. 2 and 19). In postabsorptive dogs, physiological
changes in plasma glucagon have been shown to alter hepatic glucose
production through changes in both glycogenolysis and gluconeogenesis
(2, 3). The dose-response relationships between glucagon and hepatic
glycogenolysis and gluconeogenesis appear to be similar (33), but the
time courses of the responses are different. The gluconeogenic effect
of glucagon is initially small, whereas the glycogenolytic effect is
marked (2, 3, 35). Thereafter, the gluconeogenic effect of glucagon on
the liver increases progressively while glucagon-induced glycogenolysis decreases (2, 3, 35). It is possible, therefore, that the increase in
glycogenolysis caused by glucagon limits the initial gluconeogenic
effects of the peptide and that the decline in glycogenolysis over time
allows gluconeogenesis to increase.
BAY R 3401 is a novel compound that reduces blood glucose levels by
inhibiting glycogen phosphorylase in the liver after oral administration (unpublished data). The active metabolite of the compound, BAY U 6751, inhibits the a-
and b-forms of the enzyme with an in
vitro 50% inhibitory concentration of 55 and 19 ng/ml, respectively.
The aim of the present study, therefore, was to assess the effects of
inhibiting glycogenolysis (with BAY R 3401) on basal and
glucagon-stimulated gluconeogenesis in the conscious dog.
 |
MATERIALS AND METHODS |
Animals and surgical procedures.
Experiments were performed on 10 overnight-fasted mongrel dogs
(17.4-29.0 kg, mean ± SE 22.4 ± 1.1 kg) of either sex that had been fed a standard meat and chow diet (34% protein-46%
carbohydrate-14% fat-6% fiber based on dry wt; Kal Kan, Vernon, CA,
and Purina Lab Canine Diet no. 5006, Purina Mills, St. Louis, MO) once
daily. 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 the Vanderbilt University Medical Center
Animal Care Committee. At least 16 days before an experiment, a
laparotomy was performed under general endotracheal anesthesia (15 mg/kg body wt pentobarbital sodium presurgery and 1.0% isoflurane as
an inhalation anesthetic during surgery), and catheters for blood
sampling were placed into a femoral artery, the portal vein, a hepatic
vein, a jejunal vein, and splenic vein as previously described (7, 8,
11, 28, 29, 31, 33, 35, 37). The catheter for drug infusion was placed
into the stomach as previously described (29). On the day of the
experiment, the catheters were exteriorized under local anesthesia (2%
lidocaine; Abbott, North Chicago, IL), their contents were aspirated,
and they were flushed with saline. Angiocaths (20 gauge; Abbott) were inserted into both cephalic veins for infusion of indocyanine green,
radioactive tracers, and glucose and into a saphenous vein for the
infusion of somatostatin.
On the day before the experiment, the leukocyte count and hematocrit
were determined. Dogs were used for an experiment only if they had
1) a leukocyte count
<18,000/mm3,
2) a hematocrit >38%,
3) a good appetite, and
4) normal stools.
Experimental design.
Each experiment consisted of a 100-min tracer and dye equilibration
period (
140 to
40 min), a 40-min control period
(
40 to 0 min), and two 90-min experimental periods (0 to 180 min). A priming dose of
[3-3H]glucose (41.7 µCi) was given at
140 min. Continuous infusions of
[3-3H]glucose (0.34 µCi/min),
[U-14C]alanine (0.67 µCi/min), and indocyanine green (0.1 mg · m
2 · min
1)
were also started at
140 min and were continued throughout the
experiment. At
140 min a peripheral infusion of somatostatin (0.8 µg · kg
1 · min
1)
was started to inhibit endogenous insulin and glucagon secretion. Intraportal replacement infusions of insulin (0.25 mU · kg
1 · min
1)
and glucagon (0.6 ng · kg
1 · min
1)
were started simultaneously with initiation of the somatostatin infusion. The plasma glucose level was then monitored every 5 min, and
the rate of insulin infusion was adjusted until the plasma glucose
level was stabilized at a euglycemic value. Once stabilization had been
achieved, the insulin infusion rate was left unchanged. The final
infusion rates of insulin used in placebo and drug were 0.26 and 0.24 mU · kg
1 · min
1,
respectively. Two experimental protocols were used. An intragastric bolus of a 0.5% methylcellulose-saline solution (50 ml) with (10 mg/kg) (drug group) or without (placebo group) BAY R 3401 was given at
0 min. After a 90-min test period, the infusion rate of glucagon was
increased fourfold in both groups for another 90 min.
Analytic procedures.
Plasma glucose concentrations and plasma glucose radioactivity
(3H and
14C) were determined as previously
described (4, 35). Plasma [14C]alanine and
[14C]lactate specific
activities were determined with the use of short-column, ion-exchange
chromatography as previously described by Chiasson et al. (4). Blood
concentrations of lactate, alanine, glycerol, ketones, glutamine, and
glutamate and plasma concentration of nonesterified fatty acids were
determined according to the methods reported previously (7, 8, 29, 35).
Individual blood amino acid levels were assessed with the use of the
method of Venkatakrishnan et al. (34) with an interassay
coefficient of variation (CV) of 4%. Plasma arterial and hepatic vein
indocyanine green concentrations were determined spectrophotometrically
at 805 nm (23).
Liver samples were obtained at the end of the experiments by
euthanizing the dog with pentobarbital sodium, exposing the liver by
laparotomy, and freeze clamping ~5-g liver sections from each lobe.
The time elapsed from euthanasia to freeze clamping was <4 min. The
entire liver was then removed from the dog and weighed. The frozen
liver samples were stored at
70°C for subsequent analysis. On the day of the assay, samples were powdered and homogenized, and
glycogen concentrations were determined as described previously (18).
Net incorporation of 3H and
14C into glycogen was determined
after liquid scintillation counting of the processed samples.
Immunoreactive plasma insulin, glucagon, and cortisol and plasma
epinephrine and norepinephrine were determined as previously described
(35).
Materials.
[3-3H]glucose (NEN,
Boston, MA) was used as the glucose tracer (500 µCi/0.005 mg), and
[U-14C]alanine
(Amersham, Chicago, IL) was used as the labeled gluconeogenic precursor
(171 mCi/mmol). Indocyanine green was purchased from Hynson, Westcott,
and Dunning (Baltimore, MD) and was prepared in sterile water. Insulin
was obtained from Squibb-Novo (Princeton, NJ), and glucagon was
obtained from Eli Lilly (Indianapolis, IN). Cyclic somatostatin was
purchased from Bachem (Torrance, CA). The insulin, glucagon, and
somatostatin infusates were prepared with normal saline and contained
3% (vol/vol) of the dog's own plasma. Cortisol radioimmunoassay kits
were obtained from Micromedic Systems (Horsham, PA).
Calculations.
Hepatic blood flow was assessed by measuring hepatic extraction of
indocyanine green (23). Based on data from Greenway and Stark (17), the
proportions of the hepatic blood supply provided by the hepatic artery
and portal vein were assumed to be 28 and 72%, respectively. This
ratio conforms to data that we obtained with Doppler flow probes during
pancreatic clamps (31). These proportions were assumed to remain
constant throughout all experiments, since treatment did not
significantly affect hepatic blood flow. Net hepatic substrate balance
was calculated using the formula [H
(0.28A + 0.72P)] × HF, where A, P, and H are the arterial, portal
vein, and hepatic vein substrate concentrations, respectively, and HF
is the hepatic blood or plasma flow. When blood levels of the substrate
were measured, blood flow was used in the calculation, whereas plasma
flow was used when plasma levels were measured. Tracer-determined
glucose production and glucose utilization were determined by the
method of DeBodo et al. (10) and with the use of the two-compartment
model of Mari (25). The data calculated by the two-compartment model
were described. The
[14C]glucose
production rate was determined using the tracer technique as described
by Chiasson et al. (4).
The hepatic gluconeogenic conversion rate of alanine to glucose and the
efficiency of the hepatic gluconeogenesis were calculated by dividing
the [14C]glucose
production rate
[disintegrations · min
1
(dpm) · kg
1 · min
1]
by the specific activity of alanine (dpm/µmol) entering the liver
(using weighted arterial and portal specific activities) and by the
rate of net hepatic
[14C]alanine uptake,
respectively.
[14C]lactate was
considered in the gluconeogenic calculations (both gluconeogenic
conversion and efficiency) only when
[14C]lactate was
consumed by the liver. In the case of conversion, the specific activity
of the precursor pool was estimated by taking into account the relative
contribution of
[14C]lactate uptake
and [14C]alanine
uptake by the liver. These contributions were used to determine the
average specific activity of these precursors entering the liver. The
conversion rate and efficiency are actually minimal estimates of
gluconeogenesis because of dilution of the gluconeogenic precursor
specific activity within the hepatic oxalacetate pool and the fact that
they assess gluconeogenesis from only two precursors. To bracket the
true gluconeogenic rate, a maximal estimate can be obtained by assuming
that all of the gluconeogenic precursors taken up by the liver are
completely converted to glucose, whereas a minimal estimate can be
determined by multiplying the maximal estimate by the tracer-determined
gluconeogenic efficiency. To use this method, we assumed that the net
hepatic uptake of pyruvate was 10% of the net hepatic lactate uptake
rate (36). As reported by McGuinness et al. (28), the kidney is not a
net consumer of alanine, although this organ is capable of
gluconeogenesis. Although the kidney is a net consumer of lactate, the
amount of 14C radioactivity in
lactate relative to that of alanine was small (
10%). Therefore,
[14C]glucose
production by the kidney had little, if any, impact on the calculation
of gluconeogenic efficiency.
Hepatic glycogen content was determined by multiplying the glycogen
concentration (mg/g of liver) by liver weight. The amount of glycogen
synthesized from glucose (the direct pathway) was calculated by
dividing 3H radioactivity
incorporated into liver glycogen by the average 3H specific activity in arterial
plasma glucose between 0 and 180 min. The amount of
[14C]glucose deposited
in glycogen from circulating glucose was determined by multiplying the
mass (mg) of glycogen deposited in the liver by the average
14C specific activity of arterial
plasma glucose between 0 and 180 min. The amount of
[14C]glucose in
glycogen arising from gluconeogenesis (indirect pathway) was calculated
by subtracting the
[14C]glucose deposited
from circulating glucose from the total
[14C]glucose in the
liver. The amount of glycogen deposition from gluconeogenesis was
determined by dividing the radioactivity deposited via gluconeogenesis
by the average
[14C]lactate specific
activity in the hepatic vein (presumed to reflect the intrahepatic
pyruvate specific activity) between 0 and 180 min.
Evaluation of inhibitory effect of BAY R 3401 on
hepatic glycogen phosphorylase activity.
To confirm the effect of BAY R 3401 on glycogenolysis, eight male
Sprague-Dawley rats (310-320 g) were fasted for 24 h and divided
into two groups. Two hours after oral administration of a 0.5%
methylcellulose-saline solution (0.5 ml) with (10 mg/kg) (drug group)
or without BAY R 3401 (placebo group), rats were anesthetized with
pentobarbital sodium (50 mg/kg body wt, ip). Venous blood was drawn
from the external jugular vein, and the major liver lobe was frozen in
situ with aluminum tongs precooled in liquid nitrogen. Glycogen
phosphorylase activity was analyzed according to the method of Golden
et al. (14).
Two hours after oral administration of the drug or the vehicle to rats,
the blood glucose level was lower in drug group (70.2 ± 0.9 mg/dl)
than in placebo group (80.3 ± 2.0 mg/dl). Phosphorylase a and total phosphorylase activities
(nmol · mg
protein
1 · min
1)
were decreased by the drug treatment (7.0 ± 1.9 vs. 14.3 ± 3.1 and 17.3 ± 3.9 vs. 31.1 ± 2.9), respectively.
These data confirm the ability of BAY R 3401 to suppress glycogen
breakdown.
Statistical analysis.
Data are expressed as means ± SE. Statistical comparisons were made
with the use of two-way analysis of variance with repeated-measures design. Post hoc analysis was performed with the use of the paired t-test or the unpaired
t-test (32).
 |
RESULTS |
Hormone levels and hepatic blood flow.
Portal and arterial glucagon levels were basal and unchanged in both
protocols during the first two periods. They rose approximately threefold, however, when the glucagon infusion rate was increased (Fig.
1). Portal and arterial insulin levels
remained basal and unchanged throughout the study with both placebo and
drug administration (Fig. 1). Arterial cortisol (2.2 ± 0.2 in
placebo and 2.0 ± 0.4 µg/ml in drug), norepinephrine (174 ± 37 in placebo and 122 ± 21 pg/ml in drug), and epinephrine (96 ± 36 in placebo and 90 ± 25 pg/ml in drug) levels did not
change with either treatment. Hepatic blood flow was initially higher
in drug group (31 ± 2 ml · kg
1 · min
1)
than in the placebo group (21 ± 2 ml · kg
1 · min
1),
but liver blood flow did not change over time or with treatment in
either group.

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Fig. 1.
Arterial and portal plasma levels of insulin and glucagon before and
during a 4-fold increase in intraportal glucagon brought about in
presence of basal insulin with or without intragastric administration
of BAY R 3401 (drug) in 18-h-fasted conscious dogs. Animals received
intragastric bolus injection of vehicle or BAY R 3401 (10 mg/kg) at 0 min. Data are means ± SE for 5 experiments.
+ Significantly different
from control period in identical group
(P < 0.05).
|
|
Glucose kinetics.
The plasma glucose level fell slightly in the placebo group before the
rise in glucagon while net hepatic glucose output (NHGO) remained
unchanged (Fig. 2). With drug
administration, NHGO decreased markedly (
1.5
mg · kg
1 · min
1
by 90 min; P < 0.05),
necessitating glucose infusion (Table 1) to
maintain euglycemia. In the placebo group the rise in glucagon infusion
increased NHGO by 4.0 ± 0.5 mg · kg
1 · min
1
in 10 min (P < 0.05). NHGO then fell
but remained elevated relative to the previous period. As a result of
the increase in NHGO, the plasma glucose level rose from 90 ± 3 to
171 ± 18 mg/dl by the end of the study
(P < 0.05). The rise in glucagon in
the drug treatment group increased NHGO by 2.0 ± 0.6 mg · kg
1 · min
1
within 10 min (P < 0.05). NHGO then
quickly returned to the rate evident in the previous period. As shown
in Table 1, glucose had to be infused in the drug-treated group to
maintain euglycemia, even during the period of increased glucagon.
Rates of tracer-determined endogenous glucose production
(Ra), utilization
(Rd), and clearance (Cl)
remained unchanged in the placebo group (Table
2) until glucagon increased, at which time
Ra rose markedly (by 3.7 ± 0.4 mg · kg
1 · min
1
at 10 min; P < 0.05),
Rd increased by 40%
(P < 0.05), and Cl fell slightly
(not significant,
10%). In the drug treatment group Ra fell from 2.5 ± 0.2 to 1.3 ± 0.2 mg · kg
1 · min
1
(P < 0.05) in the first test period,
whereas Rd and Cl did not change.
The rise in glucagon increased endogenous
Ra by 1.4 ± 0.3 mg · kg
1 · min
1
at 10 min (P < 0.05), after which it
fell. Rd and Cl increased slightly
(Table 2; 10%; P < 0.05).

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Fig. 2.
Arterial plasma glucose levels and changes in net hepatic glucose
balance before and during a 4-fold increase in intraportal glucagon
brought about in presence of basal insulin with or without intragastric
administration of BAY R 3401 (drug) in 18-h-fasted conscious dogs.
Animals received intragastric bolus injection of vehicle in placebo and
of BAY R 3401 (10 mg/kg) at 0 min. Net hepatic glucose outputs before
placebo and drug administration were 1.4 ± 0.1 and 2.0 ± 0.1 mg · kg 1 · min 1,
respectively. Animals treated with BAY R 3401 received glucose
peripherally (Table 2) from 0 min on to establish and maintain
euglycemia. Values for arterial plasma glucose are means ± SE and
for changes in net hepatic glucose balance are mean differences ± SE from mean value of net hepatic glucose balance in control period.
* Significantly different from corresponding value in placebo
group (P < 0.05).
+ Significantly different
from control period in identical group
(P < 0.05).
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Table 1.
Rate of glucose infusion necessary to maintain euglycemia with
intragastric administration of BAY R 3401
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Arterial lactate and alanine levels and net hepatic
balances.
As shown in Fig. 3, treatment with placebo
did not cause a change in either the arterial lactate level or net
hepatic lactate balance. In the presence of placebo the rise in
glucagon was associated with an increase in net hepatic lactate
production (by 6.7 ± 6.5 µmol · kg
1 · min
1
by 10 min), although this change, unlike the rise in the blood lactate
level (P < 0.05), did not reach
significance. In contrast, drug administration caused net hepatic
lactate output to cease (although, once again, the change was not
significant) and stimulated net hepatic lactate uptake of 3.5 ± 1.0 µmol · kg
1 · min
1,
despite a fall in the arterial lactate level (567 ± 154 to 374 ± 70 µM; P < 0.05) before
glucagon infusion. The rise in glucagon in the treatment group was
associated with a further increase in net hepatic lactate uptake to 7.3 ± 0.8 µmol · kg
1 · min
1
(P < 0.05) and a further decrease in
the arterial lactate level (to 290 ± 21 µM by 180 min;
P < 0.05). The lactate level was
reduced by >90% in response to the drug treatment, and the liver
consumed rather than produced lactate
(P < 0.05).

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Fig. 3.
Arterial blood lactate levels and changes in net hepatic lactate
balance before and during a 4-fold increase in intraportal glucagon
brought about in presence of basal insulin with or without intragastric
administration of BAY R 3401 (drug) in 18-h-fasted conscious dogs.
Animals received intragastric bolus injection of vehicle in placebo and
of BAY R 3401 (10 mg/kg) at 0 min. Net hepatic lactate balances before
placebo and drug treatment were 0.66 ± 1.75 and 2.42 ± 3.71 µmol · kg 1 · min 1,
respectively. Values for arterial plasma lactate levels are means ± SE and for changes in net lactate balance are mean differences ± SE
from mean value of net hepatic lactate balance in control period.
* Significantly different from corresponding value in placebo
group (P < 0.05).
+ Significantly different
from control period in identical group
(P < 0.05).
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As shown in Fig. 4, treatment with placebo
did not affect the arterial blood alanine level, net hepatic alanine
uptake, or net hepatic alanine fractional extraction. In the presence
of placebo the rise in glucagon did not change the arterial alanine level significantly, but it caused small increases in net hepatic alanine uptake (2.6 ± 0.5 to 3.4 ± 0.4 µmol · kg
1 · min
1;
P < 0.05) and the net hepatic
fractional extraction of alanine (0.36 ± 0.04 to 0.50 ± 0.05;
P < 0.05) by 180 min. With drug
administration alanine metabolism did not change significantly until
the glucagon concentration rose. The rise in glucagon induced a fall in
the arterial alanine level (from 256 ± 40 to 168 ± 15 µM;
P < 0.05) and increases in net
hepatic alanine uptake (from 3.3 ± 0.6 to 3.8 ± 0.5 µmol · kg
1 · min
1)
and fractional extraction (from 0.39 ± 0.04 to 0.62 ± 0.06; P < 0.05). The latter was twofold
greater than that seen in the absence of drug.

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Fig. 4.
Arterial blood alanine level and changes in net hepatic alanine uptake
and in hepatic alanine fractional extraction before and during a 4-fold
increase in intraportal glucagon brought about in presence of basal
insulin with or without intragastric administration of BAY R 3401 (drug) in 18-h-fasted conscious dogs. Animals received intragastric
bolus injection of vehicle in placebo or of BAY R 3401 (10 mg/kg) at 0 min. Net hepatic alanine uptakes before placebo and drug administration
were 2.5 ± 0.3 and 2.9 ± 0.2 µmol · kg 1 · min 1,
respectively. Hepatic alanine fractional extractions before placebo and
drug administration were 0.34 ± 0.04 and 0.31 ± 0.05, respectively. Values for arterial blood alanine levels are means ± SE and for changes in net alanine uptake and alanine fractional
extraction are mean differences ± SE from mean value of each
parameter in control period. * Significantly different from
corresponding value in placebo group
(P < 0.05).
+ Significantly different
from control period (P < 0.05).
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Arterial glycerol levels and net hepatic
balance.
Arterial blood glycerol levels, net hepatic glycerol uptake, and net
hepatic fractional glycerol uptake remained unchanged with placebo
treatment and were unaffected by the fourfold rise in glucagon (Fig.
5). Drug administration caused an increase
in net fractional extraction of glycerol by the liver from 0.56 ± 0.05 to 0.66 ± 0.05 by 90 min (P < 0.05), but neither the arterial glycerol level nor net hepatic
glycerol uptake changed significantly. In the presence of drug the rise
in glucagon resulted in increases in the net hepatic uptake (from 1.4 ± 0.3 to 1.8 ± 0.3 µmol · kg
1 · min
1)
and fractional extraction of glycerol by the liver (from 0.66 ± 0.06 to 0.81 ± 0.03) by 150 min. These
changes were significantly greater than those seen in the absence of
drug.

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Fig. 5.
Arterial blood glycerol level and changes in net hepatic glycerol
uptake and in hepatic glycerol fractional extraction before and during
a 4-fold increase in intraportal glucagon brought about in presence of
basal insulin with or without intragastric administration of BAY R 3401 (drug) in 18-h-fasted conscious dogs. Animals received intragastric
bolus injection of vehicle or of BAY R 3401 (10 mg/kg) at 0 min. Net
hepatic glycerol uptakes before placebo and drug administration were
1.15 ± 0.13 and 0.32 ± 0.28 µmol · kg 1 · min 1,
respectively. Hepatic glycerol fractional extractions before placebo
and drug administration were 0.63 ± 0.03 and 0.57 ± 0.04, respectively. Values for arterial blood glycerol are means ± SE and
for changes in net hepatic glycerol uptake and hepatic glycerol
fractional extraction are mean differences ± SE.
* Significantly different from corresponding value in placebo
group (P < 0.05).
+ Significantly different
from control period in identical group
(P < 0.05).
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Arterial levels and net hepatic balances of the
gluconeogenic amino acids.
Arterial glutamine, glycine, serine, and threonine levels and their
uptakes by the liver remained unchanged with placebo or drug treatment
before glucagon infusion (Table
3). The rise in glucagon
shifted net glutamine balance from production to uptake and resulted in
a decrease in blood glutamine levels in both groups (P < 0.05). The blood levels of
glycine, serine, and threonine fell in response to glucagon in both
groups. Their rates of uptake by the liver were not significantly
changed but their net hepatic fractional extractions increased
significantly (P < 0.05).
Gluconeogenic parameters.
Net hepatic gluconeogenic substrate uptake, obtained by summing the net
hepatic uptakes of lactate, glycerol, pyruvate, and all gluconeogenic
amino acids, did not change in the placebo group before or during the
period of increased glucagon (Fig. 6).
Likewise, the conversion of gluconeogenic precursors to glucose and
gluconeogenic efficiency did not increase in the placebo group (Fig.
7) before glucagon infusion, although
gluconeogenic conversion did increase in response to the rise in
glucagon. In contrast, drug administration increased net hepatic
gluconeogenic precursor uptake by 50%
(P < 0.05), and the rise in glucagon
caused a further increase up to 15 µmol · kg
1 · min
1.
Gluconeogenic conversion and efficiency did not change in response to
drug administration, and although glucagon may have increased gluconeogenic conversion slightly, the change was not significant. The
amounts of 3H and
14C incorporated into liver
glycogen by the end of study were two and five times higher,
respectively, in the presence of drug than placebo. Glycogen content at
180 min was significantly higher with drug (46.7 ± 0.5 mg/g liver)
than placebo (30.9 ± 5.0 mg/g liver). Glycogen synthesis via both
the direct and indirect pathways was also higher with drug (66.6 ± 5.5 and 48.6 ± 9.7 µmol glucosyl U/kg body wt) than placebo (41.1 ± 5.1 and 11.3 ± 1.7 µmol glucosyl U/kg body wt)
(P < 0.05).

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Fig. 6.
Net hepatic gluconeogenic precursor uptake before and during a 4-fold
increase in intraportal glucagon brought about in presence of basal
insulin with or without intragastric administration of BAY R 3401 (drug) in 18-h-fasted conscious dogs. Animals received an injection of
vehicle in placebo or of BAY R 3401 (10 mg/kg). Rate was obtained by
summing net hepatic uptake rates of lactate, glycerol, pyruvate, and
all gluconeogenic amino acids. Lactate uptake was considered zero when
lactate was released by liver. Values are means ± SE for 5 experiments. GNG, gluconeogenesis. * Significantly different from
corresponding value in placebo group
(P < 0.05).
+ Significantly different
from control period (P < 0.05).
|
|

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Fig. 7.
Gluconeogenic conversion of
[14C]alanine and
[14C]lactate to
[14C]glucose and
intrahepatic efficiency of this process before and during a 4-fold
increase in intraportal glucagon brought about in presence of basal
insulin with or without intragastric administration of BAY R 3401 (drug) in 18-h-fasted conscious dogs. Animals received intragastric
bolus injection of vehicle or BAY R 3401 (10 mg/kg) at 0 min. Data are
means ± SE.
+ Significantly different
from control period in identical group
(P < 0.05).
|
|
Arterial nonesterified fatty acid and ketone body
levels and net hepatic balance.
Arterial levels, net hepatic uptake, and hepatic fractional extraction
of nonesterified fatty acids (NEFA) did not change significantly with placebo or drug treatment before the rise in glucagon (Fig. 8). The glucagon increment
did not alter NEFA levels or uptake in the placebo group. In the drug
treatment group, the rise in glucagon increased the net hepatic uptake
and fractional extraction of NEFA slightly
(P < 0.05). The arterial level and hepatic production of ketone bodies were not affected by placebo or
drug treatment (Fig. 9). The rise in
glucagon increased ketone body production in the presence of drug but
not placebo (Fig. 9).

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Fig. 8.
Arterial plasma nonesterified fatty acid (NEFA) levels and changes in
net hepatic NEFA uptake and in hepatic NEFA fractional extraction
before and during a 4-fold increase in intraportal glucagon brought
about in presence of basal insulin with or without intragastric
administration of BAY R 3401 (drug) in 18-h-fasted conscious dogs.
Animals received intragastric injection of vehicle in placebo or of BAY
R 3401 (10 mg/kg). Net hepatic NEFA uptakes before placebo and drug
administration were 2.03 ± 0.33 and 2.61 ± 0.5 µmol · kg 1 · min 1,
respectively. Hepatic NEFA fractional extractions before placebo and
drug administration were 0.18 ± 0.02 and 0.17 ± 0.01, respectively. Values are means ± SE for arterial plasma NEFA levels
and are mean differences ± SE from mean value of net hepatic NEFA
uptake and of hepatic fractional extraction in control period.
* Significantly different from corresponding value in placebo
group (P < 0.05).
+ Significantly different
from control period in identical group
(P < 0.05).
|
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Fig. 9.
Arterial blood ketone body levels and changes in net hepatic ketone
body production before and during a 4-fold increase in intraportal
glucagon brought about in presence of basal insulin with or without
intragastric administration of BAY R 3401 (drug) in 18-h-fasted dogs.
Animals received intragastric injection of vehicle in placebo or of BAY
R 3401 (10 mg/kg). Net hepatic ketone body productions before placebo
and drug administration were 1.23 ± 0.2 and 1.85 ± 0.39 µmol · kg 1 · min 1,
respectively. Values are means ± SE for arterial ketone body levels
and are mean differences ± SE from mean value of net hepatic ketone
body uptake and of hepatic fractional extraction in control period.
BOHB, 3-hydroxybutylic acid; AC, acetoacetic acid.
+ Significantly different
from control period in identical group
(P < 0.05).
|
|
 |
DISCUSSION |
The relative contributions of glycogenolysis and gluconeogenesis to
basal glucose production in the present study were the same as those
noted in previous studies (2, 3, 7, 35). Gluconeogenic efficiency
increased slightly with time in the placebo group (Fig. 7; Table
4), but this observation agrees with our earlier data (7). Whether it represents a transition of the animal into
a more fasted state or the attainment of tracer equilibration in the
metabolite pools of the liver remains unclear (7). The magnitudes of
the increased glucose output, lactate production, and gluconeogenic
efficiency induced by hyperglucagonemia in the placebo group (Figs. 2,
3, 7) were similar to those observed in previous studies (33, 35). The
early glucagon-stimulated increase in glucose production must have
resulted from an increase in glycogenolysis, because the initial
increments in the net uptake of gluconeogenic precursors and
gluconeogenic efficiency were very small (Table 4). Glycogenolysis was
responsible for at least 80% of the overall increase in glucose
production (88% at 100 min and 77% at 180 min) (Table 4). A
time-dependent decrease in the glycogenolytic response to glucagon has
been reported previously (3), and its cause has been discussed (3). It
can thus be concluded that the placebo (intragastric administration of
vehicle) produced no metabolic effects of its own.
Drug treatment, on the other hand, decreased basal hepatic glucose
production by 70% and limited the glucagon-induced increase in glucose
production by 76%. Whereas plasma glucose level was maintained at
basal levels during the period of increased glucagon in the
drug-treated group, the rise in glucagon elevated plasma glucose level
in the placebo group (Fig. 2). Because hyperglycemia has been shown to
limit net hepatic glycogenolysis (2), the effect of drug treatment on
the glycogenolytic action of glucagon is even more pronounced than is
evident from a comparison of the glucose production rates in the two
groups.
It is also evident that drug treatment increased the net uptake of
gluconeogenic precursors by the liver (Fig. 6; Table 4). As a result,
the maximal contribution of gluconeogenesis to glucose production
increased to 80% before the rise in glucagon and was 91% by the end
of the glucagon infusion period. Tracer-determined whole body glucose
production reflects both hepatic and renal glucose production (1, 28).
In the overnight-fasted dog, the kidney has been shown to produce
glucose at rates equal to 13% (28) or 24% (1) of tracer-determined
whole body glucose production. With the assumption that renal glucose
production, which represents gluconeogenesis, did not change during the
treatment with the drug, glucose production at the end of the
experimental period was probably entirely due to a combination of renal
and hepatic gluconeogenesis. Thus the decreased basal and
glucagon-induced glucose production observed in the presence of BAY R
3401 was probably the result of an almost complete inhibition of
glycogenolysis.
As noted above, the decrease in glycogenolysis brought about by drug
treatment increased the basal rate of gluconeogenic precursor uptake by
the liver. It also resulted in an increase in the ability of glucagon
to stimulate net hepatic gluconeogenic precursor uptake (Fig. 6). The
latter parameter provides a maximal estimate of gluconeogenesis that
closely approximates the directly determined gluconeogenic rate (15).
The increase in the net hepatic uptake of gluconeogenic precursors
induced by decreasing glycogenolysis was not accompanied by increases
in the rates of gluconeogenic conversion or efficiency (Fig. 7).
Because glycogen synthesis operates simultaneously with glycogen
breakdown (9, 24), an inhibition of glycogenolysis can cause an
increase in the retention of 14C
from gluconeogenic precursors in glycogen. If this were the case, true
gluconeogenic efficiency would be underestimated by an amount equal to
the rate of
[14C]glucose
deposition in glycogen. Glycogen content at the end of the study was
significantly higher in the drug treatment group than in the placebo
group, and the amount of 14C
retained in glycogen after drug administration was four times that
after placebo treatment. With the assumption that
14C incorporation into glycogen
occurred constantly during the 180 min after the control period, the
rates of 14C incorporation into
glycogen in placebo and drug were equivalent to 3 and 15% of the
hepatic [14C]glucose
production rate, respectively. Even if one considers the combination of
[14C]glucose
production plus
[14C]glucose
deposition in glycogen, the reduction of glycogenolysis did not enhance
gluconeogenic efficiency. With the assumption that the
14C was incorporated into glycogen
only during the rise in glucagon, the rates of
14C incorporation into glycogen in
the placebo and in the drug treatment group corresponded to 8 and 28%
of the [14C]glucose
production rate during this period. If the
[14C]glucose
production and
[14C]glucose
deposition in glycogen are again added, gluconeogenic efficiency
increased 17% in placebo and 50% in drug during the rise in glucagon.
In this case, the effect of glucagon to increase gluconeogenic
efficiency would have increased significantly
(P < 0.05) when glycogenolysis was
inhibited. Thus we cannot rule out a change in gluconeogenic efficiency
in response to the inhibition of glycogenolysis, but even if it
occurred it was not very large.
The drug failed to increase the fractional extraction of
gluconeogenic precursors (Figs. 3-5). The rates of amino acid
uptake by the liver depend on their concentration gradient across the membrane as well as on activity or number of the amino acid
transporter(s) (12, 27). Because there is rapid equilibration between
the plasma lactate pool and the intracellular lactate and pyruvate pools (39), the increase in net hepatic lactate uptake (Fig. 3)
probably reflected a decrease in the intracellular pyruvate levels.
Decreased concentration of intracellular pyruvate within the liver
might accelerate the transamination of alanine and, as the result,
therefore, contribute to the driving force for hepatic amino acid
uptake (12). Because fatty acids have been shown in vitro to inhibit
glycolysis and increase gluconeogenesis from lactate, (30) and because
fatty acid oxidation has been reported to support the gluconeogenic
effect of glucagon (19), increased fatty acid uptake and the oxidation
might serve to augment the glucagon-induced uptake of gluconeogenic
precursors.
The rise in glucagon shifted net hepatic glutamine balance from slight
net production to net uptake (Table 3), as observed in a previous study
(18). In contrast to alanine uptake, decreasing glycogenolysis did not
affect basal hepatic glutamine balance or glucagon-stimulated net
hepatic uptake of this amino acid (Table 3). The regulation of hepatic
glutamine metabolism may differ from the regulation of alanine
metabolism, because the transporter that mediates the uptake of
glutamine (system N) is different from that of alanine (system A) (5).
In addition, glucagon stimulates intracellular glutamine degradation
via the activation of glutaminase.
Glucagon caused increases in net hepatic NEFA uptake and net hepatic
ketone production in drug-treated animals, whereas this peptide had no
such effect in the control group, in line with previous studies (22,
35). The glucagon-induced increases in NEFA uptake and ketogenesis in
the drug-treated group were not due to an increased NEFA availability,
as indicated by unchanged plasma NEFA levels and increased fractional
extraction of free fatty acids (FFA) (Fig. 8). A study by Goresky et
al. (16) in the liver of anesthetized dogs (16) in which the rate
constants for transport and metabolism of tracer
[1-14C]palmitate were
assessed by means of the multiple-indicator dilution technique
suggested that net NEFA uptake is not solely determined by the FFA load
but that intracellular factors are equally important. Wasserman et al.
(37) showed in conscious dogs that glucagon exerted a ketogenic effect
during prolonged exercise and suggested that the effect was on the
ketogenic process within the liver. In vitro studies in perfused liver
or hepatocytes have shown that glucagon stimulates triglyceride
degradation, fatty acid oxidation, and ketogenesis and that it inhibits
fatty acid synthesis (13, 26). Because lactate has an inhibitory effect
on ketogenesis in the liver (13), on the other hand, the dramatically
reduced blood lactate levels are associated with a reduction in the
direct inhibitory effects of hyperglycemia and lactate on hepatic
ketogenesis. Consequently, the effects of glucagon on inhibition of
hepatic fatty acid synthesis and stimulation of hepatic ketogenesis are more evident, and the resultant decrease in the intracellular concentration of fatty acids might in turn promote an increase in FFA
uptake.
In conclusion, the present results show that BAY R 3401 decreased
glycogenolysis in the conscious dog. Furthermore, decreased glycogenolysis per se augmented gluconeogenesis by increasing net
hepatic gluconeogenic precursor uptake and by increasing FFA metabolism
within the liver. This suggests the existence of a reciprocity between
glycogenolysis and gluconeogenesis and fatty acid metabolism in
maintaining total glucose output by the liver. The augmentation of the
gluconeogenic effect of glucagon seen when glycogenolysis was inhibited
suggests that the increase in glycogenolysis, which always follows a
rise in glucagon, delays and/or represses the gluconeogenic
effect of this peptide within the hepatocytes.
 |
ACKNOWLEDGEMENTS |
We thank Jon Hastings and the members of the Vanderbilt Diabetes
Research and Training Center Core Labs (Wanda Snead, Eric Allen, Pamela
Venson, Pat Donahue, Annapurna Venkatakrishnam, and Paul Flakoll) for
technical support.
 |
FOOTNOTES |
This research was supported by Miles Laboratories (Bayer) Grant. Part
of this work was presented at the 55th Annual Meeting of the American
Diabetes Association, Atlanta, GA, June, 1995.
Address for reprint requests: M. Shiota, Molecular Physiology and
Biophysics, Vanderbilt Univ. School of Medicine, 710E Medical Research
Bldg. 1, 21st Ave. South and Garland Ave., Nashville, TN
37232-0615.
Received 5 November 1996; accepted in final form 12 June 1997.
 |
REFERENCES |
1.
Cersosimo, E.,
R. L. Judd,
and
J. M. Miles.
Insulin regulation of renal glucose metabolism in conscious dogs.
J. Clin. Invest.
93:
2303,
1994[Medline].
2.
Cherrington, A. D.,
R. W. Stevenson,
K. E. Stein,
M. A. Davis,
S. R. Myers,
B. A. Adkins,
N. N. Abumrad,
and
P. E. Williams.
Insulin, glucagon, and glucose as regulators of hepatic glucose uptake and production in vivo.
Diabetes Metab. Rev.
3:
307-332,
1987[Medline].
3.
Cherrington, A. D.,
P. E. Williams,
G. I. Shulman,
and
W. W. Lacy.
Differential time course of glucagon's effect on glycogenolysis and gluconeogenesis in the conscious dog.
Diabetes
30:
180-187,
1981[Abstract].
4.
Chiasson, J. L.,
J. E. Liljenquist,
W. W. Lacy,
A. S. Jennings,
and
A. D. Cherrington.
Gluconeogenesis: methodological approaches in vivo.
Federation Proc.
36:
229-235,
1977[Medline].
5.
Christensen, H. N.
Role of amino acid transport and countertransport in nutrition and metabolism.
Physiol. Rev.
70:
43-77,
1990[Free Full Text].
6.
Clore, J. N.,
P. S. Glickman,
J. E. Nestler,
and
W. G. Blackard.
In vivo evidence for hepatic autoregulation during FFA-stimulated gluconeogenesis in normal humans.
Am. J. Physiol.
261 (Endocrinol. Metab. 24):
E425-E429,
1991[Abstract/Free Full Text].
7.
Connolly, C. C.,
K. E. Steiner,
R. W. Stevenson,
D. W. Neal,
P. E. Williams,
K. G. M. M. Alberti,
and
A. D. Cherrington.
Regulation of glucose metabolism by norepinephrine in conscious dogs.
Am. J. Physiol.
261 (Endocrinol. Metab. 24):
E764-E772,
1991[Abstract/Free Full Text].
8.
Connolly, C. C.,
R. W. Stevenson,
D. W. Neal,
D. H. Wasserman,
and
A. D. Cherrington.
The effects of lactate loading on alanine and glucose metabolism in the conscious dog.
Metabolism
42:
154-161,
1993[Medline].
9.
David, M.,
W. A. Petit,
M. R. Laughlin,
R. G. Shulman,
J. E. King,
and
E. J. Barrett.
Simultaneous synthesis and degradation of rat liver glycogen.
J. Clin. Invest.
86:
612-617,
1990[Medline].
10.
DeBodo, R. C.,
R. Steele,
N. Altszuler,
A. Dunn,
and
J. S. Bishop.
On the hormonal regulation of carbohydrate metabolism.
Recent Prog. Horm. Res.
19:
445-489,
1963.
11.
Diamond, M. P.,
R. C. Rolbings,
K. E. Steiner,
P. E. Williams,
W. W. Lacy,
and
A. D. Cherrington.
Effect of alanine concentration independent of changes in insulin and glucagon on alanine and glucose homeostasis in the conscious dog.
Metabolism
37:
28-33,
1988[Medline].
12.
Fabournoux, P.,
C. Rémésy,
and
C. Demigné.
Control of alanine metabolism in rat liver by transport processes or cellular metabolism.
Biochem. J.
210:
645-652,
1983[Medline].
13.
Geelen, M. J. H.,
R. A. Harris,
A. C. Beynen,
and
S. A. McCune.
Short-term hormonal control of hepatic lipogenesis.
Diabetes
29:
1006-1022,
1980[Medline].
14.
Golden, S.,
P. A. Wals,
and
J. Katz.
An improved procedure for the assay of glycogen synthase and phosphorylase in rat liver homogenates.
Anal. Biochem.
77:
436-445,
1977[Medline].
15.
Goldstein, R., B. Palmer, R. Liu, and D. Massillon. The
effects of chronic hypercortisolemia on gluconeogenesis assessed using
two independent methods in vivo.
Diabetes 44, Suppl. 1: 55A, 1995.
16.
Goresky, C. A.,
D. S. Daly,
S. Mishkin,
and
I. M. Arias.
Uptake of labeled palmitate by the intact liver: role of intracellular binding sites.
Am. J. Physiol.
234 (Endocrinol. Metab. Gastrointest. Physiol. 3):
E542-E553,
1978[Abstract/Free Full Text].
17.
Greenway, C. V.,
and
R. D. Stark.
Hepatic vascular bed.
Physiol. Rev.
51:
23-65,
1971[Free Full Text].
18.
Hendrick, G. K.,
R. T. Frizzell,
P. E. Williams,
and
A. D. Cherrington.
Effect of hyperglucagonemia on hepatic glycogenolysis and gluconeogenesis after a prolonged fast.
Am. J. Physiol.
258 (Endocrinol. Metab. 21):
E841-E849,
1990[Abstract/Free Full Text].
19.
Hue, L.
The role of futile cycles in the regulation of carbohydrate metabolism in the liver.
Adv. Enzymol. Relat. Areas Mol. Biol.
52:
278-331,
1981.
20.
Jahoor, F.,
E. J. Peters,
and
R. R. Wolfe.
The relationship between gluconeogenic substrate supply and glucose production in humans.
Am. J. Physiol.
258 (Endocrinol. Metab. 21):
E288-E296,
1990[Abstract/Free Full Text].
21.
Jenssen, T.,
N. Nurjhan,
A. Consoli,
and
J. E. Gerich.
Failure of substrate-induced gluconeogenesis to increase overall glucose appearance in normal humans. Demonstration of hepatic autoregulation without a change in plasma glucose concentration.
J. Clin. Invest.
86:
489-497,
1990[Medline].
22.
Keller, U.,
and
G. I. Shulman.
Effect of glucagon on fatty acid oxidation and ketogenesis in conscious dog.
Am. J. Physiol.
237 (Endocrinol. Metab. Gastrointest. Physiol. 6):
E121-E129,
1979[Free Full Text].
23.
Leevy, C. M.,
C. L. Mendenhall,
W. Lesko,
and
M. M. Howard.
Estimation of hepatic blood flow with indocyanine green.
J. Clin. Invest.
41:
1169-1179,
1962.
24.
Magnusson, I.,
D. L. Rothmans,
B. Jucker,
G. M. Cline,
R. G. Shulman,
and
G. I. Shulman.
Liver glycogen turnover in fed and fasted humans.
Am. J. Physiol.
266 (Endocrinol. Metab. 29):
E796-E803,
1994[Abstract/Free Full Text].
25.
Mari, A.
Estimation of the rate of appearance in the non-steady state with a two-compartment model.
Am. J. Physiol.
263 (Endocrinol. Metab. 26):
E400-E415,
1992[Abstract/Free Full Text].
26.
McGarry, J. D.,
and
D. W. Foster.
Regulation of hepatic fatty acid oxidation and ketone body production.
Annu. Rev. Biochem.
49:
395-420,
1980[Medline].
27.
McGivan, J. D.,
J. C. Ramsell,
and
J. H. Lacey.
Stimulation of alanine transport and metabolism by dibutyryl cyclic AMP in the hepatocytes from fed rats. Assessment of transport as a potential rate-limiting step for alanine metabolism.
Biochim. Biophys. Acta
644:
295-304,
1981[Medline].
28.
McGuinness, O. P.,
T. Fujiwara,
S. Murrell,
D. Bracy,
D. Neal,
D. O'Connor,
and
A. D. Cherrington.
Impact of chronic stress hormone infusion on hepatic carbohydrate metabolism in the conscious dog.
Am. J. Physiol.
265 (Endocrinol. Metab. 28):
E314-E322,
1993[Abstract/Free Full Text].
29.
Moore, M. C.,
M. J. Pagliassoti,
L. L. Swift,
J. Asher,
J. Murrell,
D. Neal,
and
A. D. Cherrington.
Disposition of a mixed meal by the conscious dog.
Am. J. Physiol.
266 (Endocrinol. Metab. 29):
E666-E675,
1994[Abstract/Free Full Text].
30.
Morand, C.,
C. Besson,
C. Demigne,
and
C. Remesy.
Importance of the modulation of glycolysis in the control of lactate metabolism by fatty acids in isolated hepatocytes from fed rats.
Arch. Biochem. Biophys.
309:
254-260,
1994[Medline].
31.
Myers, S. R.,
O. P. McGuinness,
D. W. Neal,
and
A. D. Cherrington.
Intraportal glucose delivery alters the relationship between net hepatic glucose uptake and the insulin concentration.
J. Clin. Invest.
87:
930-939,
1991[Medline].
32.
Snedecor, G. W.,
and
W. G. Cochran.
Statistical Methods (6th ed.). Ames: Iowa State Univ. Press, 1967.
33.
Stevenson, R. W.,
K. E. Steiner,
M. A. Davis,
G. K. Hendrick,
P. E. Williams,
W. W. Lacy,
L. Brown,
P. Donahue,
D. B. Lacy,
and
A. D. Cherrington.
Similar dose responsiveness of hepatic glycogenolysis and gluconeogenesis to glucagon in vivo.
Diabetes
36:
382-389,
1987[Abstract].
34.
Venkatakrishnan, A.,
M. J. Abel,
R. A. Cambell,
E. P. Donahue,
T. C. Uselton,
and
P. J. Flakoll.
Whole blood analysis of gluconeogenic amino acids for estimation of de novo gluconeogenesis using pre-column o-phthalaldehyde derivatization and high-performance liquid chromatography.
J. Chromatogr. B
676:
1-6,
1996[Medline].
35.
Wada, M.,
C. C. Connolly,
C. Tarumi,
D. W. Neal,
and
A. D. Cherrington.
Hepatic denervation does not significantly change the response of the liver to glucagon in conscious dogs.
Am. J. Physiol.
268 (Endocrinol. Metab. 31):
E194-E203,
1995[Abstract/Free Full Text].
36.
Wahren, J.,
P. Felig,
G. Ahlborg,
and
L. Jorfeldt.
Glucose metabolism during leg exercise in man.
J. Clin. Invest.
50:
2713-2725,
1971.
37.
Wasserman, D. H.,
J. A. Spalding,
D. Bracy,
D. B. Lacy,
and
A. D. Cherrington.
Exercise-induced rise in glucagon and ketogenesis during prolonged muscular work.
Diabetes
38:
799-807,
1989[Abstract].
38.
Winkler, B.,
I. Rathgel,
R. Steele,
and
N. Altszler.
Conversion of glycerol to glucose in the normal dog.
Am. J. Physiol.
219:
497-502,
1970[Medline].
39.
Wolfe, R. R.,
F. Jahoor,
and
H. Miyoshi.
Evaluation of the isotopic equilibration between lactate and pyruvate.
Am. J. Physiol.
254 (Endocrinol. Metab. 17):
E532-E535,
1988[Abstract/Free Full Text].
40.
Youn, J. H.,
and
R. H. Bergman.
Enhancement of hepatic glycogen by gluconeogenic precursors: substrate flux or metabolic control?
Am. J. Physiol.
258 (Endocrinol. Metab. 21):
E899-E906,
1990[Abstract/Free Full Text].
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