Lack of hepatic "interregulation" during inhibition
of glycogenolysis in a canine model
K.
Fosgerau1,
S. D.
Mittelman2,
A.
Sunehag3,
M. K.
Dea2,
K.
Lundgren1, and
R. N.
Bergman2
1 Department of Diabetes Biochemistry and Metabolism, Novo
Nordisk, DK-2760 Maaloev, Denmark; 2 Department
of Physiology and Biophysics, University of Southern California,
Los Angeles, California 90033; and 3 Children's Nutrition
Research Center, Houston, Texas 77030
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ABSTRACT |
It has been proposed that the
glycogenolytic and gluconeogenic pathways contributing to endogenous
glucose production are interrelated. Thus a change in one source of
glucose 6-phosphate might be compensated for by an inverse change in
the other pathway. We therefore investigated the effects of
1,4-dideoxy-1,4-imino-D-arabinitol (DAB), a potent glycogen
phosphorylase inhibitor, on glucose production in fasted conscious
dogs. When dogs were treated acutely with high glucagon, glucose
production rose from 1.93 ± 0.14 to 3.07 ± 0.37 mg · kg
1 · min
1
(P < 0.01). When dogs were treated acutely with DAB in
addition to high glucagon infusion, the stimulation of the
glycogenolytic rate was completely suppressed. Glucose production rose
from 1.85 ± 0.20 to 2.41 ± 0.17 mg · kg
1 · min
1
(P < 0.05), which was due to the increase in
gluconeogenesis from 0.93 ± 0.09 to 1.54 ± 0.08 mg · kg
1 · min
1
(P < 0.001). In conclusion, infusion of DAB inhibited
glycogenolysis; however, the absolute contribution of gluconeogenesis
to glucose production was not affected. These results suggest that
inhibition of glycogenolysis could be an effective antidiabetic treatment.
type 2 diabetes; glycogen phosphorylase; 1,4-dideoxy-1,4-imino-D-arabinitol
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INTRODUCTION |
THE PRODUCTION OF
GLUCOSE by liver and kidney provides requisite fuel to the brain
and is therefore under complex regulation (4, 14). Thus,
under a wide variety of circumstances such as fasting (14)
or exercise (4), the blood glucose level is valiantly
defended despite extreme variability in metabolic need. This fine
regulation is aberrant in type 2 diabetes, which is characterized by
peripheral insulin resistance and chronic hyperglycemia. The observed
hyperglycemia after overnight fasting has been ascribed to a failure to
suppress endogenous glucose production (EGP) in the face of peripheral
insulin resistance (12, 13), implying defect(s) in the
regulation of hepatic gluconeogenesis (GNG) and/or glycogenolysis (GLY)
(11, 26, 27). Additionally, impairment of insulin's
ability to suppress EGP after a meal is at least partially responsible
for the impaired glucose tolerance observed in type 2 diabetes
(13).
The role of hormonal (28, 36) and metabolite control on
GNG and glycogen degradation in liver has been well studied. However, the relative roles of GNG and GLY in liver remain controversial, because, for technical reasons, GNG has proven difficult to quantify (28, 36). Further confounding the understanding of GNG and GLY is the apparent compensatory interrelationship ("hepatic
interregulation") between these two pathways (19, 21, 22, 24,
31, 39). Thus basal EGP remained constant when GNG was acutely
increased by infusion of gluconeogenic precursors (19, 21,
22) or was inhibited with ethanol (24, 31). In
rats, this interregulatory effect was demonstrated despite markedly
decreased concentrations of liver glycogen. Collectively, these data
suggest that an initial modification of the gluconeogenic rate is
followed by compensatory changes in the glycogenolytic rate, thus
maintaining a constant EGP and satisfying the energetic needs of the
central nervous system.
Despite the potentially crucial role that intracellular hepatic
interregulation may play in controlling glycemia in health and
diabetes, little is known regarding the mechanism of this phenomenon
(19, 21, 22, 24, 31, 39). Possible sites of
interregulation include coordinated modification of glycogen and/or
gluconeogenic enzyme activities and/or pathway regulation by glucose
6-phosphate or other key intermediates. Furthermore, it is currently
not clear whether the role of hepatic interregulation is associated
with modulation of GNG only or represents a general mechanism that
maintains a glucose output proportional to need regardless of which
pathway(s) is affected.
One approach to determine whether the intraregulation phenomenon is
independent of which pathway is affected is to study whether specific
suppression of GLY leads to a compensatory increase in GNG. This was
done in the present study by using
1,4-dideoxy-1,4-imino-D-arabinitol (DAB), a novel, potent,
and specific inhibitor of hepatic glycogen phosphorylase (GP), whose
effects have been shown both in vitro and in vivo (1, 17).
The drug was infused in overnight-fasted (11 h), conscious mongrel dogs
under basal and glucagon-stimulated conditions. GNG was measured using
the dideuterated water method of Kalhan et al. (23) and
Landau et al. (25). We asked whether or not a specific
reduction in glycogen degradation in liver would result in an equal and
opposite increase in GNG to maintain constant EGP. In a similar study,
Shiota et al. (34) reported that inhibition of GLY
enhanced glucagon-stimulated gluconeogenic precursor uptake by the
liver of conscious dog. Their findings are discussed.
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METHODS |
Animals.
Eight conscious male mongrel dogs (25.1 ± 3.8 kg) were studied
according to Principles of Laboratory Animal Care (National Institutes of Health Publication no. 85-23, revised 1985) and California law. Animals were housed under controlled kennel conditions (12:12-h light-dark cycle) in the Keck Medical School (University of
Southern California) Vivarium. Animals had free access to standard chow
(25% protein, 9% fat, 49% carbohydrate, and 17% fiber; Wayne Dog
Chow, Alfred Mills, Chicago, IL) and tap water. Animals were used for
an experiment only if they had a hematocrit >38%, a good appetite,
and normal body temperature and stools. Food was withdrawn 11 h
before experiments. Chronic catheters were implanted
7 days before
the experiments, as previously described (32). One
catheter was placed in the portal vein 4 cm upstream from the porta
hepatis for portal infusions of insulin and glucagon. Slow infusions at this site are equally distributed among the lobes of the liver (6). A second catheter was placed in the femoral vein and
advanced to the inferior vena cava for the infusion of tracer,
somatostatin, and DAB. A third catheter was placed in the jugular vein
for the sampling of mixed venous blood. On the morning of each
experiment, a catheter was acutely inserted into the saphenous vein for
the variable infusion of glucose.
Experimental protocol.
Each dog underwent four protocols under euglycemic clamps performed in
random order. The protocols were separated by 3 wk to clear
2H2O from the circulation. At
t =
240 min, a bolus of 2H2O
(10 g/kg) was injected into the femoral vein to achieve a 1%
enrichment of body water. D-[3-3H]glucose was
given as a primed, continuous infusion beginning at t =
150 min (25 µCi prime + 0.25 µCi/min infusion; NEN Research Products, Du Pont, Boston, MA). Also at this time, a femoral infusion of somatostatin (1 µg · kg
1 · min
1; Bachem,
Torrance, CA) was started to suppress endogenous insulin and glucagon
release, and basal portal replacement infusions of insulin (porcine
insulin: 0.3 mU · kg
1 · min
1; Novo
Nordisk, Copenhagen, Denmark) and glucagon (porcine glucagon: 1.3 ng · kg
1 · min
1; Sigma
Chemical, St. Louis, MO) were begun. Basal samples were taken at
t =
240 (denoting the fasting concentration) and
150 min (denoting the basal concentration). Blood samples were drawn every 15 min from t =
150 to
30 min, and glucose
was measured on-line at these and all later time points. Glucose was
clamped at basal (t =
150 min) values by a variable
infusion of glucose (Glcinf) labeled with
D-[3-3H]glucose (2.7 µCi/g) to minimize
large changes in the specific activity (16). After tracer
equilibration, blood samples were drawn at t =
30,
20,
10, and 0 min. At t = 0 min, DAB dissolved in
saline or saline vehicle was given as a primed, continuous infusion for
210 min (1.943 mg/kg prime + 0.0187 mg · kg
1 · min
1 infusion,
Novo Nordisk). The selected dose of DAB (5.87 mg/kg) was based on
preliminary experiments in dogs (K. Lundgren and L. Ynddal, unpublished
observations) to obtain a constant plasma concentration of DAB.
The full pharmacokinetic profile of DAB is currently not known and is a
subject for further investigation. At t = 60 min, the
dogs were infused portally with high glucagon (GN; 5 ng · kg
1 · min
1) or 0.9%
saline (SAL) in addition to the basal replacement concentrations of
insulin and glucagon. Thus each animal (n = 8) was
subjected to four protocols: DAB + GN, SAL + GN, DAB + SAL, and SAL only. Mixed venous blood samples for assays were collected
every 5 min from t = 0 to t = 30 min,
followed by collections every 10 min to t = 60 min,
again every 5 min from t = 60 to t = 90 min, again every 10 min to t = 120 min, and every 15 min from t = 120 to t = 180 min.
Finally, blood samples were obtained at t = 180, 190, 200, and 210 min. All solutions were infused at a flow rate of 0.25 ml/min, except for DAB, which was infused at 0.2175 ml/min. Samples for
determination of plasma glucose, tritiated glucose, glycerol, lactate,
insulin, and glucagon concentration were collected in 50 µl of EDTA
(2 g/100 ml for 1.5 ml blood) in tubes coated with lithium fluoride and
heparin. Trasylol (aprotinin: 75 µl/ml blood; Miles, Kankakee, IL)
was added to samples for glucagon measurement to inhibit proteolysis of
the hormone. Samples for the measurement of free fatty acids (FFA) were
collected in EDTA with paraoxon to suppress lipoprotein lipase
(40). Samples were centrifuged immediately in a vacuum
centrifuge, and the plasma was separated into microcentrifuge tubes.
Plasma samples were either kept on ice and processed the same day or
stored at
80°C until assayed. For comparison of rates of GNG, GLY,
and EGP, we defined four periods as follows: period 1 (P1),
30 to 0 min; period 2 (P2), 40-60 min; period
3 (P3), 90-110 min, and period 4 (P4),
180-210 min. The outline of the infusion protocol is shown in Fig.
1.

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Fig. 1.
Time schedule and protocol for injections and infusions.
Dogs were fasted overnight for 11 h before onset of the protocol.
At t = 210 min, fasting blood samples were taken, a
bolus of 2H2O (10 g/kg) was given, and the
experiment was started. At t = 150 min, basal blood
samples were taken, and a primed infusion of
[3-3H]glucose was started. In addition, infusion of
somatostatin (SRIF) and replacement concentrations of glucagon and
insulin were initiated at t = 150. A primed
1,4-dideoxy-1,4-imino-D-arabinitol (DAB) or saline (SAL)
infusion was initiated at t = 0 min, and infusion of
high concentrations of glucagon (GN) or SAL was initiated at
t = 60 min. Period 1 (P1) was defined from
t = 30 to 0 min; period 2 (P2) from
t = 40 to 60 min; period 3 (P3) from
t = 90 to 110 min, and period 4 (P4) was
defined from t = 180 to 210 min. Experiments were
concluded at t = 210 min. See METHODS for
further details.
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Assays.
Glucose and lactate were measured with a YSI 2700 autoanalyzer (Yellow
Springs Instrument, Yellow Springs, OH) immediately after sampling.
Glycerol was measured with a 912 Hitachi analyzer (Boehringer Mannheim,
Mannheim, Germany). Samples for
D-[3-3H]glucose were deproteinized with
Ba(OH)2 and ZnSO4, and supernatants were
evaporated in a vacuum, reconstituted in water, and counted in Ready
Safe scintillation fluid (Beckman liquid scintillation counter; Beckman
Instruments, Fullerton, CA). Tracer infusates were processed in the
same manner. FFA were measured with a kit from Wako (NEFA C; Wako Pure
Chemical Industries, Richmond, VA). Glucagon was assayed using a kit
obtained from Linco Research (St. Louis, MO). Insulin was measured by
ELISA, on the basis of two murine monoclonal antibodies that bind to
different epitopes on the insulin molecule (2). Novo
Nordisk provided materials for the insulin assay, including the dog
standard. Deuterium incorporation at carbon-6 (C6) of glucose was
determined using the hexamethylenetetramine derivative, as described by
Kalhan et al. (23) and Landau et al. (25) and
modified by A. Sunehag (unpublished observation). This method
measures GNG from pyruvate and underestimates GNG by the contribution
from glycerol and to some extent by a potential, incomplete
equilibration between the deuterium enrichment in body water and at
glucose C6 (23, 25). The conclusions from the present
studies are based on comparisons among experiments. Thus a potential,
incomplete equilibration of the deuterium enrichment is unlikely to
differ among the experiments. Furthermore, to evaluate potential
differences in the contribution from glycerol, glycerol concentrations
were measured in all experiments. Total GNG can be determined from the
deuterium enrichment at glucose C5; however, this is an extremely
tedious and time-consuming method. The deuterium enrichment in
hexamethylenetetramine was analyzed by gas chromatography-mass spectrometry (HP6890/5973; GC column: HP 5: 25 m × 0.25 mm × 1.0 µm, Hewlett-Packard, Palo Alto, CA) in the electron impact
mode with selected monitoring of mass-to-charge ratios 140 and 141. Hexamethylenetetramine enrichments were converted to the corresponding glucose enrichments by use of a standard curve prepared from
[1-2H]glucose (99 atom % 2H, Cambridge
Isotope Laboratories, Andover, MA) after conversion to sorbitol
(30). Deuterium enrichment in body water (represented by
plasma water) was measured by isotope ratio mass spectrometry (Finnigan
Delta-E, Finnigan MAT, San Jose, CA) after reduction to hydrogen gas
according to accepted methods (37, 38).
Calculations.
EGP was calculated using Steele's model (32). During
periods 1 and 4, the specific activity of glucose
(SAGlc) and the concentration of measured hormones and
metabolites were stable. Periods 2 and 3 were
included for a comparison of glucose kinetics just before GN was
started (P2) and when the new level of plasma glucagon was reached (P3)
for a closer examination of the proposed interregulatory mechanism.
Estimates of EGP in the latter two periods were based on the assumption
of stable concentrations of metabolites and SAGlc.
GNG-to-EGP ratios can be calculated as the deuterium bound to C6 of
glucose divided by plasma 2H2O content when
steady state is achieved (23, 25). A recent study in
preterm human babies indicates that the deuterium-C6 method provides an
accurate method of quantifying GNG, so long as glycerol GNG is not
included (35). In the present study, a correction
factor was necessary, because the glucose pool was diluted by a
variable infusion of exogenous glucose, which did not affect the
deuterium enrichment of plasma water. The correction factor was
calculated on the basis of a single compartment model (see
APPENDIX). The rate of GLY was calculated as (EGP
GNG).
Statistical analysis.
All results are given as means ± SE. Balanced ANOVA was used to
test for the effects of DAB and glucagon on all dog outcome variables.
One dog did not complete the DAB + GN and SAL + GN protocols;
this dog was otherwise similar to the rest of the group. To allow
performance of a balanced ANOVA, the average values from the remaining
seven dogs were substituted for the missing values. When significance
was reached by ANOVA, a paired Student's t-test was used to
compare P1 and P4. This paired Student's t-test was based
on n = 7, or n = 8 when possible, and
did not include estimated data.
 |
RESULTS |
Hormone replacement.
The outline of the protocol is shown in Fig. 1. During P1, the average
concentration of plasma glucagon of 45 ± 6 ng/l (Fig. 2) matched the fasting concentration
(Table 1). Although glucagon concentration remained unchanged in the absence of GN [Fig. 2: 41 ± 6 vs. 45 ± 6 ng/l, P4 vs. P1, P = nonsignificant (NS)], infusion of glucagon at 5 ng · kg
1 · min
1 tripled
plasma glucagon to 134 ± 8 ng/l during P4 (Fig. 2,
P < 0.001). DAB infusion did not affect glucagon
concentration in any of the protocols (P = NS).
Infusion of insulin (0.3 mU · kg
1 · min
1)
underreplaced the fasting and basal concentrations (Tables 1 and
2, P < 0.001). Plasma
concentrations of FFA and glycerol during the experiments were similar
to basal concentrations (Tables 1 and 2, P = NS) but
were modestly reduced compared with fasting concentrations (Tables 1
and 2, P < 0.001).

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Fig. 2.
Summary of plasma concentrations of glucagon. Dogs were
infused with basal replacement concentrations of glucagon (1.3 ng · kg 1 · min 1) throughout
the experiment or basal replacement followed (at t = 60 min) by GN (5 ng · kg 1 · min 1); see
METHODS for details. No effect of DAB on plasma glucagon
concentrations was observed. Vertical bars indicate SE values.
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We observed no effects of DAB or hyperglucagonemia on plasma insulin,
glycerol, FFA, or lactate concentrations (Table 2, P = NS). Deuterium enrichment of plasma water was stable during P1-P4 in
all protocols, whereas the deuterium enrichment at C6 of
glucose increased slightly during P4 (Fig.
3). Blood levels of glucose and
glucose infusion rates (Glcinf) are shown in Fig. 4.

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Fig. 3.
Effect of DAB and GN on the deuterium enrichments of body water
(left) and carbon 6 (C6) of plasma glucose
(right). Four protocols were performed on each dog: DAB + GN (n = 7), SAL + GN (n = 7),
DAB + SAL (n = 8), and SAL only (n = 8), and the deuterium enrichment of body water and C6 of glucose was
measured during the 4 study periods for the determination of
gluconeogenesis (GNG), as described in METHODS. Deuterium
enrichment of plasma water and C6 of glucose was stable in all periods
(P1-P4) in all protocols. Vertical bars indicate SE values.
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Fig. 4.
Plasma levels of glucose and rates of glucose infusion. Plasma glucose
levels (left) and glucose infusion rates
(Glcinf; right) are shown. Plasma glucose
levels were clamped at basal (t = 150 min) values by
variable infusions of glucose (Glcinf), as described in
METHODS. Four protocols were performed on each dog:
DAB + GN (n = 7), SAL + GN (n = 7), DAB + SAL (n = 8), and SAL only (Control;
n = 8). GN caused a transient increase above the
clamped level; however, no differences in plasma glucose levels were
observed between any groups when P1 and P4 were compared. Vertical bars
indicate SE values.
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Glucose turnover.
With basal hormone replacement (control), EGP demonstrated a modest
decline during the experiment (Fig. 5),
declining from 1.79 ± 0.18 to 1.66 ± 0.17 mg · kg
1 · min
1, from P1 to
P4. This decline was due to falling GLY, which was reduced by 30% over
the experiment (Table 3,
P = NS).

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Fig. 5.
Effect of DAB and GN on the rate of endogenous glucose
production (EGP). Four protocols were performed on each dog: DAB + GN (n = 7), SAL + GN (n = 7),
DAB + SAL (n = 8), and SAL only (n = 8), and dynamic changes in EGP were measured as described in
METHODS. DAB impaired the effect of glucagon on the rate of
EGP (P < 0.05, n = 7). Vertical bars
indicate SE values.
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As expected, GN stimulated a brisk increase in glucose production,
which almost doubled from 1.93 to 3.71 mg · kg
1 · min
1 during P3.
There was then a slight evanescence in EGP, which declined modestly to
3.07 mg · kg
1 · min
1 during
P4. The glucagon stimulation of EGP was largely due to an increase in
GLY by 151% during P3.
DAB caused a small, transient decrease in EGP, which was reduced from
the control during P2 and P3. This decrease reflected a reduction in
basal GLY due to DAB. However, in the absence of glucagon stimulation,
this effect disappeared during P4. In contrast, DAB had a marked effect
to reduce EGP in the presence of glucagon: during P3, when
glucagon-stimulated EGP was maximal, there was no increase at all in
GLY in the presence of DAB + GN (0.84 ± 0.05 vs. 0.92 ± 0.24 mg · kg
1 · min
1 at
basal). Thus DAB completely inhibited the glucagon-stimulated increase
in GLY. However, because GLY fell continually at a slow rate during the
experiment with SAL alone, it is probable that, even with DAB present,
glucagon had a small effect on GLY during the experiment, because this
decline was prevented, suggesting that the infusion protocol used for
DAB did not lead to full inhibition of GP.
In contrast to GLY, the stimulation of GNG due to glucagon was
virtually the same in the presence of DAB as with GN alone (Table 3,
P2: 0.85 vs. 0.86; P3: 1.26 vs. 1.29; P4: 1.54 vs. 1.44). Also in the
nonstimulated situation, we saw no effect of DAB on GNG (Table 3, P2:
0.83 vs. 0.88; P3: 0.88 vs. 0.87; P4: 1.04 vs. 1.09).
Thus the presence of the inhibitor DAB severely suppressed GLY, as
expected, but had no measurable effect on GNG with or without GN (Table
3 and Fig. 6). In other words, hepatic
interregulation did not appear to occur under these conditions of
glycogenolysis blockade.

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Fig. 6.
Effect of DAB and GN on the rate of gluconeogenesis (GNG)
and glycogenolysis (GLY) during P4. Four protocols were performed on
each dog: DAB + GN (n = 7), SAL + GN
(n = 7), DAB + SAL (n = 8), and
SAL only (n = 8), and dynamic changes in the rate of
GLY (top) and GNG (bottom) were measured as
described in METHODS. Total EGP = GNG + GLY. GN
stimulated GLY (*P < 0.05, n = 7) and
GNG (***P < 0.001, n = 7). Additional
infusion of DAB suppressed this effect of glucagon on GLY without a
compensatory increase of GNG. DAB alone had no effect on either GNG or
GLY during P4 (P = NS, n = 8). Vertical
bars indicate SE values.
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DISCUSSION |
Despite the central role of the liver in the regulation of blood
glucose in health and diabetes, questions continue to exist regarding
the regulation and relative roles of the two pathways contributing to
EGP, GNG, and GLY (19, 24, 31, 39). When GNG was inhibited
with ethanol in patients with type 2 diabetes (31) or with
rats (24) or when GNG was acutely increased by infusion of
gluconeogenic precursors (19, 21, 22), hepatic glucose
output remained constant, suggesting that an initial modification of
the gluconeogenic rate is followed by compensatory changes in the
glycogenolytic rate. This mechanism has been termed hepatic autoregulation (19, 21, 22, 24, 31, 39). Here, however, we
prefer the term interregulation, because the term autoregulation has
traditionally been used to describe the mechanism by which the blood
glucose controls the overall hepatic glucose output (29).
Currently, it is not clear whether the suggested interregulation is a
phenomenon solely associated with modulation of GNG or a general
mechanism that maintains a constant EGP regardless of which pathway is
affected. Shiota et al. (34) reported that inhibition of
GLY by BAY R 3401 enhanced glucagon-stimulated gluconeogenic precursor
uptake by the liver of conscious dog. Also, maximal estimate of GNG was
higher in the drug group than in placebo, whereas the authors observed
no differences when comparing minimal estimates of GNG.
Here, using the compound DAB, a novel inhibitor of GP and glycogen
breakdown (1, 17), we report no changes in GNG upon inhibition of GLY with or without glucagon stimulation. The difference in findings might be explained as a deposition of gluconeogenic carbon
as glycogen, which would not be detected by the dideuterated water
method that we used in our study. Thus DAB had no influence on glycogen
synthesis in primary hepatocytes (1) or on lactate deposition into glycogen in the perfused rat liver (K. Fosgerau, N. Westergaard, and J. Breinholt, unpublished observations). In contrast, the amount of glycogen synthesized from gluconeogenic carbon with infusion of BAY R 3401 in dogs was higher in the drug group than in the placebo group (34). Alternatively, the
difference in findings might be explained as a difference in the
mechanism of action between compounds DAB and BAY R 3401. Thus, in
hepatocytes, DAB had no effect on either protein phosphorylase 1, the
enzyme responsible for dephosphorylation of GPa to GPb, or on
phosphorylase kinase (1), whereas BAY R 3401 promoted
dephosphorylation of GP (3).
Notably, maximal estimate of GNG in the study of Shiota et al.
(34) was obtained by assuming that all of the
gluconeogenic precursors taken up by the liver were completely
converted to glucose. However, a significant amount of the
gluconeogenic precursors was actually deposited as glycogen. Because
the amount of glycogen synthesized from gluconeogenic carbon was higher
in the drug group than in the placebo group (34), the
maximal estimate of GNG is relatively overestimated in the former group.
The basal rate of EGP in dogs treated with DAB was compared with that
in dogs not treated with DAB (Fig. 5). At t = 10, 15, 20, and 50 min, the rate of EGP was lower in dogs treated with DAB
(P < 0.05). This is believed to be the result of an
inhibition of basal GLY, because the average rates of GNG were not
affected by DAB during P2. However, there was a tendency toward an
increase in EGP immediately before the infusion of GN at 1 h.
Also, in the absence of GN infusion, GLY during P4 was equal in dogs
treated with DAB compared with controls (Fig. 6), suggesting that the infusion protocol used for DAB did not lead to full inhibition of GP.
Alternatively, increased lysosomal hydrolysis of glycogen may diminish
the effect of DAB on glycogen breakdown, because DAB is a weak
inhibitor of mammalian glucosidases (1) and 10% of the
hepatic glycogen is located within the lysosomes (18).
Glucagon is recognized as a critical physiological regulator of EGP and
basal glucose homeostasis (7-10, 15, 33). Elevated concentrations of glucagon have stimulating effects on both the gluconeogenic and glycogenolytic pathways, albeit following different time courses (9, 33). Thus a sustained elevated glucagon concentration causes a rapid and marked activation of GLY followed by a
declining rate of this pathway despite a sustained period of GN
["evanescent" effect (9, 33)], just as we observed (Table 3). In contrast, the rate of GNG is slowly upregulated with
elevated concentrations of glucagon, and this effect is sustained (9, 33). It is possible that the initial marked increase in GLY observed with elevated concentrations of glucagon limits glucagon's stimulatory effect on GNG at first and that the rate of GNG
subsequently increases only as GLY declines (33). The existence of such a mechanism would further support the existence of
the proposed hepatic interregulatory mechanism.
As expected, elevated glucagon had a transient stimulatory effect on
GLY followed by a slow increase in GNG in the present study (Table 3).
However, when DAB was given before glucagon, the glucagon stimulation
of EGP was sharply reduced. This inhibitory effect of DAB was sustained
throughout the experiment, as demonstrated by the fact that EGP above
basal in dogs infused with DAB + GN was only 36% of the increment
in EGP observed in animals treated with glucagon alone. We observed no
differences in plasma glucose levels between any groups when P1 and P4
were compared (Fig. 3, P = NS). However, there was a
tendency toward an increased plasma glucose level in response to
SAL + GN infusion during P3. This response in plasma glucose can
be ascribed to the effect of glucagon on GLY (7-10, 15,
33). The glucose level was restored (P4) at the clamp level by
adjustment of Glcinf (Fig. 3) and as a result of an
increased rate of disposal (Rd) (data not shown). With
infusion of DAB + GN, we observed increased plasma glucose level
during P4, which can be ascribed to the effect of glucagon on GNG
(7-10, 15, 33). Thus, judging from the enrichment
data of C6 of glucose (Fig. 4), there was a tendency toward increasing
deuterium incorporation during P4 after a GN infusion, indicating that
GNG was, in fact, increasing at this stage and that the maximum rate of
GNG was not reached in the length of the study period. The compensatory adjustment of Glcinf resulted in the observed glucose
level, which was above, but not statistically different from, the P1
level. However, it cannot be excluded that the metabolism of DAB may cause a relief of the observed GLY inhibition during P4. Previous studies in enzyme preparations of GP (17) and in the
perfused liver (K. Fosgerau, H. Westergaard, and J. Breinholt,
unpublished results) revealed no effect of glucose on the
inhibitory action of DAB.
GNG was measured during four periods (P1-P4) by means of deuterium
labeling of glucose secondary to infusion of deuterated water
(23, 25). This method is a straightforward approach to
measuring GNG; however, it underestimates GNG by the contribution from
glycerol and potentially by an incomplete equilibration between the
deuterium enrichment in body water and at C6 of glucose. In the present
study, there were no differences with regard to plasma concentrations
of glycerol and FFA between the experiments, indicating that DAB did
not affect lipolysis or glycerol delivery from adipose tissue. Thus it
is very unlikely that DAB had any effect on the gluconeogenic
contribution from glycerol. Deuterium labeling of glycogen would lead
to an overestimation of GNG; however, in the present study, deuterated
water was given during fasting conditions, where labeling of glycogen
is believed to have no significance because glycogen synthase is not
activated (20, 29). Furthermore, it was previously shown
that DAB has no effect on glycogen synthesis in primary hepatocytes
(1) or in the perfused rat liver (K. Fosgerau, H. Westergaard, and J. Breinholt, unpublished observations). Consequently, it is not believed that the choice of method for estimating GNG had any impact on our conclusions regarding GNG.
Because GNG was equal with or without DAB, the lower glucose production
observed with DAB can be ascribed to an inhibition of
glucagon-stimulated GLY. Thus, as shown in Fig. 6, DAB inhibited glucagon-stimulated GLY by 46% during P4, confirming the mechanism of
action of DAB and again demonstrating that it is effective in vivo
(1, 17). Also, because GNG in our hands was not affected by infusion of DAB regardless of the concentration of glucagon, it is
apparent that, in the fasting normal dog, hepatic interregulation may
not exist for GNG being regulated by GLY. Thus this suggests that
hepatic interregulation is a phenomenon for which GNG may alter the
rate of GLY, but not the converse (19, 21, 22, 24, 31,
39). Of course, we did not alter the rate of GNG in the present
study, so that the existence of the hepatic interregulation phenomenon
overall is not disproved by the present results, in that changes in GNG
may indeed alter GLY. Also, at the concentration of DAB used,
glucagon-stimulated GLY was not completely inhibited during P4. It
remains possible that inhibition of GLY must be more pronounced for an
effect to be seen on GNG. Finally, the possibility continues to exist
that the apparent constancy of EGP might be an extrahepatic phenomenon.
Our studies were carried out under clamped conditions. It remains a
possibility that the plasma glucose itself (20, 29), other
blood-borne compounds such as FFA (5, 23, 28), or the
central nervous system may be important extrahepatic regulators of
liver function that guarantee an appropriate delivery rate of glucose
to the peripheral tissues regardless of the balance between GNG and
GLY. Also, we measured total EGP and did not separate out the relative
roles of liver vs. kidney in glucose production. Possibly, a decrease in hepatic GNG was followed by an increase in kidney GNG; however, this
latter possibility appears unlikely, because glucose production by the
kidney provides only a minor fraction of total GNG for blood glucose
homeostasis after an overnight fast (14).
In conclusion, DAB inhibited glucagon-stimulated EGP via inhibition of
GLY but did not affect the absolute contribution of GNG to EGP with or
without glucagon stimulation. These data do not support an effect of
changing GLY on GNG. However, it is still possible that the changes in
GNG can affect the glycogenolytic rate, suggesting that the
interrelationship between glycogen degradation and GNG in controlling
EGP is complex. Because the glucagon challenge is regarded as a model
of type 2 diabetes, these data suggest that inhibition of glycogen
phosphorylase might prove beneficial in the treatment of this disease.
 |
APPENDIX |
In the present study, an undeuterated exogenous glucose infusion
(Glcinf) was utilized to clamp plasma glucose at basal
concentrations. Deuteration enrichment in body water is represented by
the deuterium enrichment in plasma (23, 25). Thus
Glcinf will dilute only the deuterium enrichment of glucose
from GNG at the C6 position but not the deuterium enrichment in body
water. The equations described by Kalhan et al. (23) and
Landau et al. (25) result in estimates of GNG as a
fraction of EGP. Because Glcinf was variable throughout the
experiments, including the "steady-state" periods, calculating GNG
as a fraction of EGP requires correction for this variable infusion
rate. A detail of the approximated corrections for Glcinf
dilution follows.
In plasma, the proportion of glucose from Glcinf must be
calculated so that, by subtraction, the proportion of EGP derived from
GNG may be determined. A one-compartment model of glucose kinetics was
utilized to calculate these proportions
|
(A1)
|
|
(A2)
|
where dGlcinf P(t)/dt is the
derivative of plasma glucose from Glcinf(t),
Glcinf(t) is the exogenous glucose infusion
rate, Rd Glc inf(t) is the rate of
disappearance of glucose from Glcinf, dEGPP(t)/dt is the derivative of
plasma glucose from EGP, EGP(t) is the rate of appearance of
endogenous glucose based on [3-3H]glucose dilution, and
Rd EGP(t) is the rate of disposal of glucose from EGP.
Rd Glc inf(t) is defined as the total glucose
uptake rate multiplied by the fraction of plasma glucose from Glcinf
|
(A3)
|
Furthermore, Rd EGP(t) is defined as the
total glucose uptake multiplied by the fraction of plasma glucose from
EGP
|
(A4)
|
Equation 3 is substituted into Eq. 1, and
Eq. 4 is substituted into Eq. 2
|
(A5)
|
|
(A6)
|
By setting an initial point for the differential equations
(Eqs. 5 and 6), the time course of
GlcinfP and EGPP may be reconstructed on
the basis of known Glcinf(t),
Rd(t), and EGP(t).
The initial point is based on two assumptions. 1) Before any
Glcinf infusion, 100% of the glucose in plasma is assumed
to originate from the EGP. 2) During the time before the
first steady-state period, changes in Glcinf were
accounted for equally by changes in Rd and EGP.
The second assumption was necessary, because the experimental protocol
required Glcinf to be infused before
[3-3H]glucose isotopic steady state, precluding the
ability to calculate Rd(t) and EGP(t)
during this period. When it was assumed that the changes in
Glcinf during this period were accounted for completely by
either EGP or Rd, the difference in EGPP was
<2%. Thus it is presumed that the GNG calculation is not highly
dependent on this assumption, and the presented results were calculated
with changes in Glcinf being accounted for equivalently by
Rd and EGP. Finally, the corrected rate of EGP was used to
calculate the rate of GNG on the basis of the equations of Kalhan et
al. (23) and Landau et al. (25), where E is
enrichment
|
(A7)
|
 |
ACKNOWLEDGEMENTS |
We are grateful to Donna Moore, Douglas Davis, Rita Thomas, Lene
Priskorn, and Elza Demirchyan for their technical expertise in handling
the dogs and in performance of assays. Also, we thank Drs. Viggo
Diness, Niels Westergaard, and James McCormack for discussions and
comments fruitful to the study.
 |
FOOTNOTES |
These studies were supported by Novo Nordisk A/S and the National
Institutes of Health (Grants DK-27619 and DK-29867). Novo Nordisk also
supported Dr. K. Fosgerau.
Present address of K. Lundgren: Zealand Pharmaceuticals A/S, Smedeland
26B, DK-2600 Glostrup, Denmark.
Address for reprint requests and other correspondence: K. Fosgerau, Pharmacological Research II, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Maaloev, Denmark (E-mail:
kf{at}novonordisk.com).
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. Section 1734 solely to indicate this fact.
Received 13 October 2000; accepted in final form 28 March 2001.
 |
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