A negative arterial-portal venous glucose gradient decreases
skeletal muscle glucose uptake
Pietro
Galassetti,
Masakazu
Shiota,
Brad A.
Zinker,
David H.
Wasserman, and
Alan D.
Cherrington
Department of Molecular Physiology and Biophysics, Vanderbilt
University School of Medicine, Nashville, Tennessee 37232-0615
 |
ABSTRACT |
The effect of a
negative arterial-portal venous (a-pv) glucose gradient on
skeletal muscle and whole body nonhepatic glucose uptake was studied in
12 42-h-fasted conscious dogs. Each study consisted of a 110-min
equilibration period, a 30-min baseline period, and two 120-min
hyperglycemic (2-fold basal) periods (either peripheral or intraportal
glucose infusion). Somatostatin was infused along with insulin (3 × basal) and glucagon (basal). Catheters were inserted 17 days
before studies in the external iliac artery and hepatic, portal and
common iliac veins. Blood flow was measured in liver and hindlimb using
Doppler flow probes. The arterial blood glucose, arterial plasma
insulin, arterial plasma glucagon, and hindlimb glucose loads were
similar during peripheral and intraportal glucose infusions. The a-pv
glucose gradient (in mg/dl) was 5 ± 1 during peripheral and
18 ± 3 during intraportal glucose infusion. The net hindlimb
glucose uptakes (in mg/min) were 5.0 ± 1.2, 20.4 ± 4.5, and
14.8 ± 3.2 during baseline, peripheral, and intraportal glucose
infusion periods, respectively (P < 0.01, peripheral vs. intraportal); the hindlimb glucose fractional
extractions (in %) were 2.8 ± 0.4, 4.7 ± 0.8, and 3.9 ± 0.5 during baseline, peripheral, and intraportal glucose infusions,
respectively (P < 0.05, peripheral
vs. intraportal). The net whole body nonhepatic glucose uptakes (in
mg · kg
1 · min
1) were 1.6 ± 0.1, 7.9 ± 1.3, and 5.4 ± 1.1 during baseline, peripheral, and
intraportal glucose infusion, respectively
(P < 0.05, peripheral vs.
intraportal). In the liver, net glucose uptake was 70% greater during
intraportal than during peripheral glucose infusion (5.8 ± 0.7 vs.
3.4 ± 0.4 mg · kg
1 · min
1).
In conclusion, despite comparable glucose loads and insulin levels,
hindlimb and whole body net nonhepatic glucose uptake decreased
significantly during portal venous glucose infusion, suggesting that a
negative a-pv glucose gradient leads to an inhibitory signal in
nonhepatic tissues, among which skeletal muscle appears to be the most
important.
hyperglycemia; arterial-portal venous glucose gradient
 |
INTRODUCTION |
AFTER THE INGESTION of a meal rich in carbohydrates,
the liver rapidly switches from net glucose production to net glucose uptake. This uptake accounts for the disposal of ~30% of the
ingested glucose. After oral glucose consumption, peak rates of net
hepatic glucose uptake (NHGU) can reach 5-8
mg · kg
1 · min
1
in both humans and dogs (1, 7, 21, 25). Both hyperglycemia and
hyperinsulinemia are known to affect the rate of NHGU; a direct linear
relationship has been described between hepatic glucose load and NHGU
both when insulin values are uncontrolled (8) and at constant
hyperinsulinemic levels (21). A significant correlation was also
observed between the plasma insulin concentration and NHGU when the
hepatic glucose load was maintained constant (20). For the complete
activation of NHGU, however, hyperglycemia and hyperinsulinemia are not
sufficient. If levels of hyperglycemia and hyperinsulinemia similar to
those observed after the ingestion of a meal are reproduced by glucose
infusion into a peripheral vein, the maximal levels of NHGU that can be
obtained are about one-third to one-half of the peak rates observed
after a meal (9, 29). Interestingly, if the same total amount of
glucose is infused directly into the portal vein, rates of NHGU similar to those observed after ingestion of glucose are observed (13). This
last situation mimics glucose ingestion, when the glucose level in the
portal vein is elevated above that in the arterial system. This
negative arterial-portal venous glucose gradient, also referred to as
the "portal signal," is now thought to trigger a third factor
that must be present, in association with hyperglycemia and
hyperinsulinemia, for the full stimulation of NHGU.
In this way, the portal signal allows the liver to discriminate between
an increased hepatic glucose load resulting from ingested as opposed to
internally produced glucose. It also tightly matches glucose absorption
from the intestine to liver glucose uptake, thereby minimizing
perturbations of glucose in the systemic circulation.
Both in the 1987 study by Adkins et al. (2) and in the 1996 study by
Pagliassotti et al. (26), the observation was made that although NHGU
increases in response to intraportal glucose delivery, whole body
glucose clearance is unchanged. This implies that during intraportal
glucose delivery, a reduction in net glucose uptake by nonhepatic
tissues must occur in parallel with the increase in NHGU. In both of
the above studies, to maintain constant the glucose load to the liver,
changes in the glucose load reaching the skeletal muscle were allowed
to occur, thus complicating data interpretation. To date, no attempt
has been made to directly identify the specific site at which the
reduction in glucose uptake caused by intraportal glucose delivery
occurs. The aim of the present study, therefore, was to determine
whether net glucose uptake by the hindlimb, which is >70% skeletal
muscle, is altered by a negative arterial-portal venous glucose
gradient.
 |
METHODS |
Animal care and surgical procedures.
Studies were performed on 20 42-h-fasted conscious mongrel dogs of
either sex, averaging 23.5 ± 1.0 kg in weight. The choice of a fast
of this duration was motivated by the metabolic state that it produces,
which closely resembles that in the overnight-fasted human (the liver
exhibits net glucose output and net lactate uptake), and by the
achievement of a stable minimum in liver glycogen content. All animals
were maintained daily on a diet of meat and chow (34% protein, 14.5%
fat, 46% carbohydrate, and 5.5% fiber based on dry wt). The animals
were housed in a facility meeting the American Association for
Accreditation of Laboratory Animal Care guidelines, and the protocol
was approved by the Vanderbilt University Animal Care Subcommittee.
Seventeen days before each study, dogs underwent a laparotomy under
general anesthesia (0.8% isoflurane). Catheters were inserted into the
right common hepatic vein, the hepatic portal vein, a common iliac
vein, and an external iliac artery as described in detail elsewhere (7,
25). Catheters were also placed in a splenic and a jejunal vein for
intraportal infusions. Doppler flow probes (Instrumentation Development
Laboratory, Baylor College of Medicine, Houston, TX) were placed around
the portal vein, the hepatic artery, and an external iliac artery. The
catheters were filled with saline containing heparin (200,000 U/l;
Abbott, North Chicago, IL); their free ends were knotted, and, together with the free ends of the Doppler leads, they were placed in a subcutaneous pocket, allowing complete closure of the incision.
Approximately 48 h before a study, blood was drawn to determine the
leukocyte count and the hematocrit of each animal. Dogs were studied
only if they had a leukocyte count
<18,000/mm3, a hematocrit
>35%, a good appetite as evidenced by consumption of all the daily
food ration, and normal stools.
On the morning of the study, the catheters and Doppler leads were
exteriorized from their subcutaneous pocket using local anesthesia (20 g/l of lidocaine; Astra Pharmaceuticals, Worcester, MA). The contents
of each catheter were aspirated, and the catheters were flushed with
saline. The splenic and jejunal catheters were used for intraportal
infusion of insulin and glucagon (Eli Lilly, Indianapolis, IN),
glucose, and p-aminohippurate (PAH;
Sigma, St. Louis, MO). PAH was used to assess the mixing of glucose in the portal and hepatic veins during intraportal glucose infusion. Portal venous, hepatic venous, common iliac venous, and external iliac
arterial catheters were used for blood sampling. On the day of study,
venous catheters (16-gauge angiocath; Deseret Medical, Becton-Dickinson, Sandy, UT) were inserted into the left cephalic vein
for [3-3H]glucose,
[U14C]glucose, and
indocyanine green infusions (ICG; Hynson, Westcott and Dunning,
Baltimore, MD); into the right cephalic vein for peripheral glucose and
PAH infusions; and into the right saphenous vein for somatostatin
infusion (Bachem, Torrance, CA). Each dog was allowed to stand quietly
in a Pavlov harness for 20-30 min before the experiment was
started.
Experimental design.
Each experiment consisted of a total of 380 min divided into a 110-min
equilibration period (from
140 to
30 min), a 30-min basal
period (baseline, from
30 to 0 min), and two 120-min
hyperglycemic periods (from 0 to 120 min and from 120 to 240 min) (Fig.
1). In all experiments, a constant infusion
of ICG (0.1 mg · m
2 · min
1) and a primed,
continuous infusion of HPLC-purified
D-[3-3H]glucose
(34-µCi bolus + 0.34 µCi/min) and
[U-14C]glucose
(20-µCi bolus + 0.20 µCi/min) were initiated at
140 min and
maintained throughout the study. At 0 min, a constant infusion of
somatostatin (0.8 µg · kg
1 · min
1)
was begun to suppress endogenous insulin and glucagon secretion, and
constant intraportal infusions of insulin (1.0 mU · kg
1 · min
1)
and glucagon (0.5 ng · kg
1 · min
1)
were established. In this way the insulin level was raised threefold, and glucagon concentration was maintained at basal values.

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Fig. 1.
Experimental design. In all animals, hyperglycemia was induced via both
peripheral and intraportal glucose infusions, but sequence of
hyperglycemic periods was reversed in one-half of dogs. ICG,
indocyanine green; @, at.
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At time = 0 min, hyperglycemia was brought about with the goal of
doubling the baseline arterial glucose concentration and maintaining it
constant until the end of the study. During the 240 min of
hyperglycemia, glucose was infused into a peripheral vein only
(peripheral glucose infusion period) for 120 min and into the portal
vein (intraportal glucose infusion period) at a constant rate (5 mg · kg
1 · min
1)
supplemented by infusion in a peripheral vein for 120 min. In all dogs,
both peripheral and portal venous infusions were given, but in one-half
of them (group A,
n = 6), the peripheral infusion was
performed first, whereas in the other one-half (group
B, n = 6), the
sequence was reversed. During peripheral glucose infusion, saline was
infused intraportally.
External iliac arterial, portal venous, hepatic venous, and common
iliac venous blood samples were taken every 15 min during the baseline
period and every 10 min during the last 30 min of both hyperglycemic
periods. The hyperglycemic clamp was monitored by means of small (0.4 ml) arterial blood samples drawn every 5 min and analyzed for glucose
within 90 s. The peripheral glucose infusion rate was adjusted as
required. The total volume of blood withdrawn did not exceed 20% of
the animal's total blood volume, and two volumes of normal saline were
infused for each volume of blood withdrawn. The methods for collecting
and processing blood samples have been described previously (18).
Analytic procedures.
Eight determinations of the plasma glucose concentration were made on
each arterial and external iliac venous plasma sample; five
determinations were made on the portal venous and hepatic venous
samples, using the glucose oxidase method on a Beckman glucose analyzer
(Beckman Instruments, Fullerton, CA). Blood concentrations of glucose
were obtained in triplicate on perchloric acid extracts using a
Technicon Autoanalyzer according to the method of Lloyd et al. (15).
Blood lactate, alanine, glycerol (perchloric acid extracts), and plasma
free fatty acid (FFA) concentrations were determined in triplicate by
enzymatic methods, using a Monarch 2000 Centrifugal Analyzer
(Instrumentation Laboratory, Lexington, MA) as previously described
(15). Plasma glucose radioactivity (3H and
14C) was determined by liquid
scintillation counting after deproteinization and evaporation to remove
3H2O
(30). Whole blood
[14C]glucose and
[14C]lactate
radioactivities were determined according to the method described by
Okajima et al. (24). Whole blood
14CO2
was liberated by acidification with hydrochloric acid and trapped on
chromatography paper with the use of hyamine hydroxide. PAH
concentrations were determined on perchloric acid extracts of blood
according to the method of Brun (6). The immunoreactive glucagon
concentration in plasma samples to which 500 KIU/ml of Trasylol (FBA
Pharmaceutical) had been added was determined with a modified version
of the method of Morgan and Lazarow (19), with an interassay
coefficient of variation (CV) of 10%. Immunoreactive insulin was
measured as previously described (19), with an interassay CV of 4%.
Calculations and data analysis.
Hindlimb blood flow was estimated using a Doppler flow probe
(Instrumentation Development Laboratory) implanted on the external iliac artery and connected to an ultrasonic, range-gated, pulsed Doppler flow meter designed by Hartley and co-workers (11, 12). Hepatic
blood flow was estimated using both ICG, according to the method of
Leevy et al. (14), and Doppler flow probes (Instrumentation Development
Laboratory) implanted on the hepatic artery and portal vein. These two
independent techniques yielded similar values. Doppler-determined
hepatic blood flow was used in data calculation for consistency with
the hindlimb measurements and because this technique provides
differential hepatic arterial and portal venous blood flow
measurements. The ICG technique, in contrast, only allows an estimate
of total hepatic blood flow and was used as a backup for the Doppler
measurements. The distribution of hepatic blood flow was 81% portal
vein and 19% hepatic artery in the baseline period and 75% portal
vein and 25% hepatic artery in the hyperglycemic periods, when
somatostatin was infused. These data are consistent with others
reported in the dog (2, 4, 20, 27), in which, although somatostatin
altered the distribution of blood flow between the portal vein and the
hepatic artery slightly, total hepatic blood flow was not significantly
affected.
When glucose is infused in the slow, laminar flow of the portal venous
circulation, mixing of the glucose in the blood can be problematic.
PAH, a substance not extracted by the liver or erythrocytes, was mixed
with the intraportal glucose infusate at a concentration that resulted
in a PAH infusion rate of 0.4 mg · kg
1 · min
1.
The recovery of PAH across the liver was measured as described previously (20, 26). The ratio between the recovery of intraportally infused PAH and the actual intraportal PAH infusion rate was calculated and used as an index of mixing of the intraportal infusate with the
blood entering and exiting the liver. Because of the magnitude of the
CV for the method of assessing PAH balance, samples were considered
statistically unmixed (>95% confidence that mixing did not occur) if
hepatic vein recovery was >140% or <60% of the actual amount of
PAH infused (20). If poor mixing, according to the above definition,
was obtained in more than one of the four time points of the
intraportal period, the animal was excluded from the study. This
occurred in eight animals. Therefore, of the 20 experiments performed,
data from only 12 were included in the reported database. In the
latter, the ratio of PAH recovery in the portal vein to the intraportal
PAH infusion rate was 0.92 ± 0.02, and the ratio of PAH recovery in
the hepatic vein to the PAH infusion rate was 0.88 ± 0.02 (a ratio
of 1.0 would represent perfect mixing). In the 12 experiments included
in the study, the infusate failed to mix with the blood <15% of the
time (7 out of 48 measurements). More importantly, when mixing errors did occur, they were random; therefore, all time points from these 12 animals were included in the database.
The hepatic substrate load was calculated directly as
where
CA and
CP represent the substrate
concentrations in arterial and portal venous blood, respectively, and
ABF and PBF represent the hepatic arterial and the portal venous blood
flows, respectively. In glucose balance calculations, plasma glucose values were converted to whole blood values by using a correction factor (CF) obtained by calculating the ratio of the whole blood glucose value to the plasma glucose value at each time point throughout the study. A separate CF was established for each sampling site for
each dog. The mean CF was 0.72 ± 0.01 in the artery, iliac vein,
and hepatic vein throughout the whole study. The CF in the portal vein
was 0.71 ± 0.01 during intraportal glucose infusion and 0.72 ± 0.01 during baseline and peripheral glucose infusion. Calculations
performed with plasma glucose values converted to blood glucose values
yielded similar results to those performed with blood glucose values
per se, but the variance was reduced because of the increased accuracy
of plasma glucose arteriovenous differences, which can be obtained
without deproteinization. The use of whole blood glucose ensures
accurate hepatic balance measurements regardless of the characteristics
of glucose entry into the erythrocyte. To circumvent any potential
errors arising from incomplete mixing of glucose in the circulation
during intraportal glucose infusion, a second, indirect method of
calculating the hepatic glucose load was used. It utilized the formula
where
GA represents the blood glucose
concentration in the artery, GIRP
represents the glucose infusion rate into the portal vein, and GUG
represents the uptake of glucose by the gastrointestinal tract. GUG was
measured during peripheral glucose infusion as the product of the
arterial-portal venous glucose difference and the portal venous blood
flow. It was considered to be the same during intraportal glucose
infusion as during the peripheral glucose infusion if the arterial
glucose concentration was identical between the two hyperglycemic
periods. When small differences in arterial glucose concentrations
between the peripheral and intraportal infusion periods were measured,
the GUG measured during peripheral glucose infusion was corrected,
based on the previously demonstrated correlation between GUG and
arterial blood glucose concentration (20), and an estimated value of
GUG during intraportal glucose infusion was thus derived. The load of
substrates exiting the liver was calculated as
where
CH is the concentration of
substrate in the hepatic vein, and HBF is the total hepatic blood flow.
Net hepatic glucose balance (NHGB) was calculated by two separate
methods as described previously (20, 27). In the first method, which
will be referred to as the direct calculation
and
in the second, referred to as the indirect calculation
In
these calculations, a positive value indicates net output. In
RESULTS, the hepatic glucose load and
NHGB shown were determined by use of the indirect calculation to be
consistent with our earlier publications. However, it should be noted
that the estimate of NHGB was similar, regardless of which method was
used in calculations. For other substrates, the direct calculation was
used to calculate net hepatic balance.
Net hindlimb substrate balances were calculated as the product of
external iliac arterial blood flow and the arteriovenous difference in
substrate concentration, as measured using samples from the external
iliac artery and common iliac vein.
For both the liver and hindlimb, substrate fractional extraction was
calculated as the ratio between net substrate uptake and substrate
load.
Whole body net nonhepatic glucose balance was calculated during the two
hyperglycemic periods as the difference between the total glucose
infusion rate and NHGU.
Rates of whole body glucose production
(Ra) and utilization
(Rd) were estimated on the basis
of [3H]glucose
specific activities, using a modification of the Steele equation as
described previously (5). During the hyperglycemic periods, endogenous
glucose production was estimated by subtracting the rate of exogenous
glucose infusion from Ra.
Rates of hindlimb glucose oxidation were calculated by dividing net
hindlimb
14CO2
production by the arterial
[14C]glucose specific
activity (corrected for
[14C]lactate specific
activity at time points at which net lactate uptake was measured). The
percentage of lactate derived from glucose across the hindlimb was
measured as the ratio of arterial
[14C]glucose to venous
[14C]lactate specific
activities.
Statistical analysis.
Because the two subgroups of six dogs underwent experimental procedures
with opposite sequences of routes of glucose infusion, the effect due
to sequence was tested using an unpaired
t-test that compared the route
differences between the groups (peripheral-portal vs. portal-peripheral
sequence). Because no difference due to sequence was detected in the
relevant variables, a "random-effect model" was then applied to
estimate the effect of the change in route of glucose infusion,
adjusting for period, time during each period, random differences in
individual animals, and, again, sequence. The results indicate that for
the relevant variables, there was no significant effect of sequence,
period, or time, although for the latter two, both peripheral and
hepatic glucose uptake showed a positive trend (i.e., going toward the
second one-half of the study, there was a slight increase in uptake; within each period, going toward a later sampling point, there was a
slight increase in uptake). The model also allowed us to estimate a
value for the difference (portal vs. peripheral glucose infusion) of a
given variable that is independent of the time point at which the
measurements are made. Once the effect of the sequence of the routes of
infusion was ruled out, data from the peripheral glucose infusion
period in all dogs and from the intraportal glucose infusion period in
all dogs were combined, and statistical comparisons were made using
paired t-tests. Differences were
considered significant at P < 0.05.
 |
RESULTS |
Hormonal levels.
The arterial plasma insulin concentration was increased by threefold
during each experimental period (Fig.
2A),
whereas the arterial plasma glucagon level was kept at a basal value
(Fig. 2B). The stability of
pancreatic hormone concentrations was demonstrated by the fact that the
CV of group mean values within each sampling period was 6% for insulin
and 1% for glucagon.

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Fig. 2.
Arterial plasma insulin (A) and
glucagon (B) concentrations in
42-h-fasted conscious dogs during baseline (euglycemia), peripheral
glucose, and intraportal glucose infusion periods (arterial plasma
glucose at ~220 mg/dl). Data are group means ± SE;
n = 12. In each dog, value from
baseline period is the mean of 3 measurements, whereas data from
peripheral and intraportal glucose infusion periods are means of 4 measurements each.
# P < 0.05 vs. baseline.
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Whole body glucose kinetics, total glucose infusion
rates, glucose levels, blood flows, and arterial-portal venous glucose
gradient.
Endogenous glucose Ra was ~2.3
mg · kg
1 · min
1
during the baseline period and was completely suppressed after the
start of exogenous glucose infusion, with no difference between the two
hyperglycemic periods. Tracer-determined glucose
Ra and endogenous glucose
Ra are shown in Table
1.
The achievement of steady state during each of the sampling periods is
evident from the stability of the total glucose infusion rates (Table
2) and the glucose concentrations in the
artery and portal, hepatic, and common iliac veins (Table 2) and of the
blood flows in the external iliac and hepatic arteries and in the
portal vein (Table 2). The arterial-portal venous glucose gradient (in
mg/dl; Fig. 3) shifted from moderately
positive during the baseline (1.9 ± 0.5) and peripheral glucose
infusion (4.6 ± 0.6) periods to markedly negative during the
intraportal glucose infusion period (
18.0 ± 3.4).

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Fig. 3.
Arterial-portal venous glucose gradient (whole blood) in 42-h-fasted
conscious dogs during baseline (euglycemia), peripheral glucose, and
intraportal glucose infusion periods (arterial plasma glucose at ~220
mg/dl). Data are group means ± SE;
n = 12. In each dog, value from
baseline period is mean of 3 measurements, whereas data from peripheral
and intraportal glucose infusion periods are means of 4 measurements
each.
# P < 0.05 vs. baseline. * P < 0.05, intraportal vs. peripheral glucose infusion.
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Hindlimb glucose metabolism and whole body net
nonhepatic glucose uptake.
The glucose load reaching the hindlimb (in mg/min; Fig.
4A)
averaged 6.6 ± 0.9 at baseline, 15.8 ± 2.1 during peripheral
glucose infusion, and 14.8 ± 2.4 during portal venous glucose
infusion (not significant vs. peripheral). The net hindlimb glucose
uptake (in mg/min; Fig. 4B) was 5.0 ± 1.2 at baseline, 20.4 ± 4.5 during peripheral glucose
infusion, and 14.8 ± 3.2 during intraportal glucose infusion
(P < 0.01 vs. peripheral).
Importantly, a similar difference in hindlimb glucose uptake between
peripheral and intraportal glucose infusion periods was observed,
independent of what route was used first. In the six dogs in which the
sequence was peripheral-portal, hindlimb glucose uptakes were 18.4 ± 3.8 and 12.4 ± 2.1 mg/min (P < 0.03); in the six dogs in which the sequence was portal-peripheral, glucose uptakes were 17.1 ± 6.2 vs. 22.4 ± 8.6 mg/min
(P < 0.05). The application of a
random-effect model of statistical analysis allowed us to estimate the
effect due to route of glucose infusion, adjusting for order of
intervention, experimental period, time within each period, and random
differences present in individual animals. The results indicate that
there is no significant effect of the order of intervention
(P = 0.94), period
(P = 0.85), or time
(P = 0.22), although the latter two
show a positive trend (i.e., hindlimb glucose uptake tended to
progressively increase toward a later phase of the study). After
adjustment for these trends, there still is a strongly significant
difference due to route of infusion (P = 0.008). The model also allowed us to estimate a value for the
difference in hindlimb glucose uptake between routes of glucose
infusion that is independent of the time point at which the
measurements are made. The estimated value was 5.9 mg/min, very close
to the measured value of 5.6 mg/min.

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Fig. 4.
Hindlimb glucose load (A), net
uptake (B), and net fractional
extraction (C) in 42-h-fasted
conscious dogs during baseline (euglycemia), peripheral glucose, and
intraportal glucose infusion periods (arterial plasma glucose at ~220
mg/dl). Data are group means ± SE;
n = 12. In each dog, value from
baseline period is mean of 3 measurements, whereas data from peripheral
and intraportal glucose infusion periods are means of 4 measurements
each.
# P < 0.05 vs. baseline. * P < 0.05, intraportal vs. peripheral glucose infusion.
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Changes in hindlimb glucose fractional extraction reflected changes in
net glucose uptake (Fig. 4C) and
were also significantly different with peripheral vs. portal glucose
infusion within each subgroup of six dogs. The decrease of this
parameter during portal venous glucose infusion resulted from a small,
nonsignificant decrease in iliac arterial blood flow along with a
marked decrease of the arteriovenous difference of glucose across the
hindlimb (7.7 ± 1.3 vs. 6.1 ± 0.8 mg/dl,
P < 0.05; Fig.
4C). The net whole body nonhepatic
glucose uptake (in
mg · kg
1 · min
1;
Fig. 5) was 1.6 ± 1.2 at
baseline and increased to 7.9 ± 1.3 (
6.3 mg · kg
1 · min
1)
during peripheral glucose. The increase over baseline was blunted by
~40% in the presence of portal glucose infusion, when it was only
5.4 ± 1.1 (
3.8 mg · kg
1 · min
1,
P < 0.05 vs. peripheral glucose
infusion). Therefore, the difference in whole body nonhepatic glucose
uptake measured between the peripheral and intraportal glucose periods
was ~2.5
mg · kg
1 · min
1.

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Fig. 5.
Net whole body nonhepatic glucose uptake in 42-h-fasted conscious dogs
during baseline (euglycemia), peripheral glucose, and intraportal
glucose infusion periods (arterial plasma glucose at ~220 mg/dl).
Data are group means ± SE; n = 12. In each dog, value from baseline period is mean of 3 measurements,
whereas data from peripheral and intraportal glucose infusion periods
are means of 4 measurements each.
# P < 0.05 vs. baseline. * P < 0.05, intraportal vs. peripheral glucose infusion.
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Of the net hindlimb glucose uptake, the amount that underwent oxidation
(in mg/min; Fig.
6A) was
0.33 ± 0.15 (5 ± 3%) at baseline, 2.18 ± 0.72 during
peripheral glucose (10.8 ± 3.5%), and 2.25 ± 0.95 during
intraportal glucose (15.4 ± 4.8%, not significant vs. peripheral
glucose). Conversely, the amount that underwent nonoxidative metabolism
(in mg/min; Fig. 6B) was 4.8 ± 1.0 at baseline, 18.2 ± 3.8 during peripheral glucose, and 12.5 ± 2.2 during intraportal glucose. Thus the decrease in hindlimb
glucose uptake reflected a change in nonoxidative glucose metabolism.

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Fig. 6.
Rates of hindlimb glucose oxidation
(A) and nonoxidative glucose
metabolism (B) in 42-h-fasted
conscious dogs during baseline (euglycemia), peripheral glucose, and
intraportal glucose infusion periods (arterial plasma glucose at ~220
mg/dl). Data are group means ± SE;
n = 12. In each dog, value from
baseline period is mean of 3 measurements, whereas data from peripheral
and intraportal glucose infusion periods are means of 4 measurements
each.
# P < 0.05 vs. baseline. * P < 0.05, intraportal vs. peripheral glucose infusion.
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Lactate, glycerol, and FFA concentrations and
hindlimb balances.
During hyperglycemia, arterial and iliac venous lactate concentrations
rose markedly (Fig.
7A); the
slightly higher increase in the arterial lactate concentration,
however, was sufficient to suppress net hindlimb lactate output
~80%, but no effect of the route of glucose infusion was observed
(Fig. 7B). The fraction of lactate
that was derived from glucose was 41 ± 7% during baseline, 62 ± 7% during peripheral glucose infusion, and 56 ± 6% during intraportal glucose infusion.

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Fig. 7.
Hindlimb lactate circulating concentrations
(A) and net output
(B) in 42-h-fasted conscious dogs
during baseline (euglycemia), peripheral glucose, and intraportal
glucose infusion periods (arterial plasma glucose at ~220 mg/dl).
Data are group means ± SE; n = 12. In each dog, value from baseline period is mean of 3 measurements,
whereas data from peripheral and intraportal glucose infusion periods
are means of 4 measurements each.
# P < 0.05 vs. corresponding baseline value.
|
|
Arterial and iliac venous glycerol concentrations were reduced by
~60% during hyperglycemia (Fig.
8A), and
a similar reduction was seen in net hindlimb glycerol output (Fig.
8B). Changes from baseline were not
altered by the route of glucose infusion. The arterial and iliac venous
FFA concentrations decreased by ~80% during hyperglycemia (Fig.
9A). Net
hindlimb FFA output decreased by ~90%. Again, no differences were
observed between the two hyperglycemic periods (Fig.
9B).

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|
Fig. 8.
Hindlimb glycerol circulating concentrations
(A) and net output
(B) in 42-h-fasted conscious dogs
during baseline (euglycemia), peripheral glucose, and intraportal
glucose infusion periods (arterial plasma glucose at ~220 mg/dl).
Data are group means ± SE; n = 12. In each dog, value from baseline period is mean of 3 measurements,
whereas data from peripheral and intraportal glucose infusion periods
are means of 4 measurements each.
# P < 0.05 vs. baseline.
|
|

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|
Fig. 9.
Hindlimb free fatty acid circulating concentrations
(A) and net output
(B) in 42-h-fasted conscious dogs
during baseline (euglycemia), peripheral glucose, and intraportal
glucose infusion periods (arterial plasma glucose at ~220 mg/dl).
Data are group means ± SE; n = 12. In each dog, value from baseline period is mean of 3 measurements,
whereas data from peripheral and intraportal glucose infusion periods
are means of 4 measurements each.
# P < 0.05 vs. baseline.
|
|
Hepatic glucose load; net glucose balance;
fractional extraction; and lactate, glycerol, and FFA hepatic
balances.
The glucose load reaching the liver (Table
3) doubled during peripheral glucose
infusion. A further small increase (~10%) occurred during
intraportal glucose infusion, reflecting the experimental design.
NHGU was observed during both hyperglycemic periods, with values 70%
higher during intraportal glucose infusion
(P < 0.05) than during peripheral
glucose infusion (Table 3). The difference in NHGU measured between the
peripheral and intraportal glucose periods was ~2.4
mg · kg
1 · min
1,
i.e., an amount almost identical to the difference in net nonhepatic glucose uptake during the two hyperglycemic periods. The fractional extraction of glucose by the liver was also higher
(P < 0.05) during intraportal
glucose infusion (Table 3), suggesting that the slightly higher hepatic
glucose load caused by the experimental design only accounted for a
small part of the difference in NHGU measured during the two
hyperglycemic periods.
Net hepatic lactate balance shifted from net uptake to net output
during both hyperglycemic periods. Net hepatic lactate output was 2.4 ± 0.8 µmol · kg
1 · min
1
higher during intraportal glucose infusion compared with peripheral glucose infusion (Table 3). Nevertheless, because its pattern of change
paralleled the changes in NHGU, net hepatic lactate output could
account for an identical percentage (~9%) of the net glucose taken
up during either glucose infusion period.
The net hepatic glycerol and FFA uptakes were decreased by ~75% and
~90%, respectively, in response to hyperglycemia. The negative
arterial-portal venous glucose gradient had no effect on their uptake
by the liver (Table 2).
 |
DISCUSSION |
The skeletal muscle is an important site of glucose removal in the
postprandial state. In resting conditions, the amount of glucose taken
up by muscle is believed to be regulated by the levels of circulating
glucose and insulin. This study sought to determine whether the portal
signal created by intraportal glucose infusion could alter hindlimb
glucose uptake. The results show that the increase in net hindlimb
glucose uptake was 57% greater when hyperglycemia was induced using a
peripheral glucose infusion than when it was created via an intraportal
glucose infusion. This effect was observed both when peripheral glucose
infusion preceded and when it followed intraportal glucose infusion,
indicating that our findings were not an artifact due to the particular
experimental design (whole body glucose uptake has been shown to
progressively increase for at least 4-5 h in
hyperglycemic-hyperinsulinemic conditions) (26). Furthermore, this
effect was obviously not limited to the hindlimb on which the
measurements were performed, as net whole body nonhepatic glucose
uptake displayed a pattern of change that reflected the changes in the
hindlimb (66% greater increase in glucose uptake during intraportal
glucose infusion). Finally, the decrease in net whole body nonhepatic
glucose uptake (2.5 mg · kg
1 · min
1)
caused by the portal signal was almost identical to the increase (2.4 mg · kg
1 · min
1)
that it caused in NHGU. Thus whole body glucose clearance was unchanged
by the route of glucose delivery. The elucidation of a molecular
mechanism responsible for the observed changes in skeletal muscle
glucose uptake is beyond the scope of this study. Among hypothetical
mechanisms, the modulation of adenosine release, as well as its
interaction with adenosine receptors or metaboreceptors, seems a
reasonable candidate, as recent evidence indicates that adenosine
inhibits glycogen breakdown in perfused rat muscle simultaneously exposed to insulin and
-adrenergic stimulation (31). The plasma concentration of FFAs has also been demonstrated to affect muscle glucose uptake via both an intracellular inhibitory effect on key
glycolytic enzymes and a direct inhibition of transmembrane glucose
transport (28). In our study, however, a similar reduction in FFAs
occurred in both hyperglycemic periods; therefore, the enhancement of
muscle glucose uptake caused by the decrease in plasma FFAs was
probably comparable in the two experimental conditions and did not
cause the observed differences in muscle glucose metabolism.
In the work of several investigators (2, 3, 20, 26), experimental
conditions were created in which the route of glucose infusion was
changed from peripheral to intraportal during hyperglycemia and
hyperinsulinemia. In all of these studies, the observation was made
that glucose uptake by extrahepatic tissues was reduced when glucose
was infused directly in the portal vein as opposed to a peripheral
vein. In 1997, Matsuhisha et al. (16) induced hyperglycemia, first via
a peripheral vein, then intraportally, and finally intraportally but
abolishing the portal signal in the cerebral vessels. A difference in
extrahepatic glucose uptake was present but was not significant between
the first two periods and became markedly significant between the
second and third periods. The lack of significance between the first
and second period may have resulted from the time-dependent increase in
muscle glucose uptake usually associated with hyperinsulinemia. It
should be noted, however, that although the reported observations
suggest a double effect of the portal signal (enhancement of net
glucose uptake by the liver and simultaneous decreases of extrahepatic glucose disposal), the above studies were focused on changes in liver
glucose metabolism. The glucose load to the liver was constant, whereas
the arterial glucose concentrations and the glucose load reaching the
hindlimb varied substantially between the portal and peripheral glucose
infusion periods. More importantly, net glucose uptake by the hindlimb,
or by any other muscle district, was never measured directly. In the
present study, the glucose load to the limb was controlled and we
assessed hindlimb glucose uptake directly. As a result, we were able to
provide strong support for the hypothesis that the reduction in
nonhepatic glucose uptake observed in response to intraportal glucose
delivery occurs primarily in skeletal muscle. The observed effect on
skeletal muscle was also specific to glucose. Net hindlimb balances of
alanine (data not shown), lactate, glycerol, and FFAs were
significantly altered by hyperglycemia but not by the route of glucose
infusion.
Our direct measurement of net skeletal muscle glucose uptake was solely
performed across one hindlimb. The metabolic behavior of the remaining
portion of the dogs' muscle mass can therefore only be hypothesized.
In 1992, Wasserman et al. (32) reported that, in the dog, the skeletal
muscle mass of a single hindlimb is 3.3% of total body weight (32).
With the consideration that our dogs averaged 23.5 kg in weight, this
would mean that 780 g of muscle accounted for the measured difference
of 5.6 mg/min in net hindlimb glucose uptake. With the assumption of a
homogenous behavior for the entire skeletal muscle mass (~45% of the
total body wt), this would finally correspond to a difference in net glucose uptake of ~76 mg/min or ~3.2
mg · kg
1 · min
1,
not far from the value of 2.5 mg · kg
1 · min
1
measured for the change in net whole body nonhepatic glucose uptake.
This overestimation by 0.7 mg · kg
1 · min
1
probably reflects the different response to hyperglycemia and hyperinsulinemia in different muscle groups. In particular, in muscle
groups that undergo tonic contraction, such as the respiratory muscles,
rates of net glucose uptake are probably mostly driven by the constant
high metabolic demand, and thus they may be influenced to a lesser
degree by the route of glucose administration.
Our experimental design did not allow the measurement of differences in
muscle glycogen content and glycogen synthase activity, as tissue
samples could only be taken at autopsy and all subjects underwent both
peripheral and intraportal glucose infusions. However, we directly
measured glucose oxidation across the hindlimb and found identical
rates during the two hyperglycemic periods. It is therefore not
unreasonable to hypothesize that most of the decrease in net skeletal
muscle glucose uptake observed during intraportal glucose infusion was
accounted for by a reduction in muscle glycogen deposition.
Nevertheless, direct evidence for this hypothesis will require
additional work.
A still unresolved issue is the definition of the nature and
characteristics of the portal signal itself. The early hypothesis of
the involvement of a humoral agent, such as a gastrointestinal peptide,
has not been supported by recent data. Conversely, progressively more
evidence is being gathered to support the possibility that the portal
signal may involve the autonomic nervous system (4, 16, 17). Both
adrenergic and cholinergic nerve terminals have been found within the
liver, with evidence for both afferent and efferent limbs (22, 23).
Further support for neural involvement has been provided by the study
of Adkins-Marshall et al. (4), in which the increase in NHGU after
intraportal glucose delivery was abolished in dogs that had undergone
complete hepatic denervation. In another recent study (16), it was
demonstrated in dogs that, if during intraportal glucose infusion
glucose was also selectively infused into the arteries reaching the
brain, leaving intact the negative glucose gradient between the rest of
the arterial blood stream and the portal vein, the increase in NHGU was
significantly blunted but not abolished. Conversely, the effect of
intraportal glucose infusion on extrahepatic glucose uptake was
completely abolished, indicating that skeletal muscle glucose uptake
can be regulated independently of glucose and insulin load through the
autonomic nervous system. A similar conclusion was drawn by Minokoshi
et al. (17), who observed an increase in glucose uptake by the skeletal
muscle in response to stimulation of the ventromedial hypothalamus.
Finally, an interesting model was proposed by Xie and Lautt (33) in
1996. These authors observed that a surgical or pharmacological
blockade of the hepatic parasympathetic nerves induced insulin
resistance in the skeletal muscle of hyperinsulinemic cats and
speculated that a factor may be released by the liver that is dependent
on intact hepatic parasympathetic nerves and that may regulate the
responsiveness of skeletal muscle to insulin. Despite these pieces of
indirect evidence, the complete elucidation of the mechanisms by which
the portal signal exerts its effect on muscle requires additional work.
In summary, when the effect of hyperglycemia resulting from intraportal
glucose delivery was compared with the effect of similar hyperglycemia
resulting from peripheral glucose delivery in the presence of identical
levels of hyperinsulinemia, not only was NHGU enhanced, but net
skeletal muscle glucose uptake was proportionally decreased. The
changes in skeletal muscle glucose metabolism do not appear to include
alterations in the rate of glucose oxidation, suggesting that a
reduction in muscle glycogen deposition is likely to be the underlying
mechanism. Our findings support the hypothesis that the portal signal
is able to direct the flow of carbons throughout the body, producing a
coordinated regulation of tissue glucose metabolism at the level of
both the liver and skeletal muscle.
 |
ACKNOWLEDGEMENTS |
We thankfully acknowledge the excellent technical assistance of
Wanda Snead and Pam Venson from the Hormone Core Laboratory of the
Vanderbilt University Diabetes Research and Training Center.
 |
FOOTNOTES |
This work was supported by National Institute of Diabetes and Digestive
and Kidney Diseases Grants R-01-DK-43706, DK-40936, and DK-50277 and
the Diabetes Research and Training Center Grant DK-20593.
Address for reprint requests: P. Galassetti, Rm. 754 MRB-I, Vanderbilt
Univ. Medical Center, Nashville, TN 37232-0615.
Received 6 October 1997; accepted in final form 13 April 1998.
 |
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