Effect of fast duration on disposition of an intraduodenal
glucose load in the conscious dog
Pietro
Galassetti,
Katherine S.
Hamilton,
Fiona K.
Gibbons,
Deanna
P.
Bracy,
Drury B.
Lacy,
Alan D.
Cherrington, and
David H.
Wasserman
Department of Molecular Physiology and Biophysics, and Diabetes
Research and Training Center, Vanderbilt University School of Medicine,
Nashville, Tennessee 37232
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ABSTRACT |
The effects of prior
fast duration (18 h, n = 8;
42 h, n = 8) on the glycemic and
tissue-specific responses to an intraduodenal glucose load were studied
in chronically catheterized conscious dogs.
[3-3H]glucose was
infused throughout the study. After basal measurements, glucose spiked
with [U-14C]glucose
was infused for 150 min intraduodenally. Arterial insulin and glucagon
were similar in the two groups. Arterial glucose (mg/dl) rose ~70%
more during glucose infusion after 42 h than after an 18-h fast. The
net hepatic glucose balance
(mg · kg
1 · min
1)
was similar in the two groups (basal: 1.8 ± 0.2 and 2.0 ± 0.3; glucose infusion:
2.2 ± 0.5 and
2.2 ± 0.7). The
intrahepatic fate of glucose was 79% glycogen, 13% oxidized, and 8%
lactate release after a 42-h fast; it was 23% glycogen, 21% oxidized, and 56% lactate release after an 18-h fast. Net hindlimb glucose uptake was similar between groups. The appearance of intraduodenal glucose during glucose infusion (mg/kg) was 900 ± 50 and 1,120 ± 40 after 18- and 42-h fasts (P < 0.01).
Conclusion: glucose administration after prolonged fasting induces
higher circulating glucose than a shorter fast (increased appearance of
intraduodenal glucose); liver and hindlimb glucose uptakes and the
hormonal response, however, are unchanged; finally, an intrahepatic
redistribution of carbons favors glycogen deposition.
fasting; glycogen; glucose uptake; gut; liver; skeletal muscle
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INTRODUCTION |
PRIOR EXERCISE that diminishes tissue glycogen stores
increases the ability of skeletal muscle to store glucose as glycogen (20, 27). This is due in part to an increase in muscle insulin sensitivity, but also in part to extramuscular adaptations (14, 20).
Intestinal glucose absorption (13), the net rate of splanchnic glucose
release (14, 20), and arterial glucose are higher (14, 18, 20) in
response to a glucose load immediately after exercise than after rest.
The signals that cause these adaptations in the postexercise state are
still unknown. It is possible that the effects of prior exercise merely
reflect the accelerated transition to a more fasted state. Prolonged
fasting can markedly affect the absorption dynamics and fate of an oral
carbohydrate load (22). One of the main determinants of the metabolic
response to food deprivation is the depletion of glycogen stores.
Hepatic glycogen is stored more rapidly in human subjects administered a glucose load after a prolonged fast as opposed to a shorter-duration fast (9, 10). Moreover, a strong positive correlation exists between
the degree of hepatic glycogen depletion and the rate of hepatic
glycogen turnover (1, 6, 21).
Despite the importance of the nutritional state in determining the
metabolic response to feeding, very little is known about how
splanchnic tissues adapt to substrate depletion induced by a prolonged
fast. It was the aim of this study to determine whether, like prior
exercise, a prolonged fast causes adaptations in the gut and liver that
facilitate the disposition of an intraduodenal glucose load. To assess
this, hepatic glycogen was depleted by prolonged fasting (42 h) to a
degree similar to that observed after prolonged moderate exercise.
Glucose fluxes and metabolism were quantified in 18- and 42-h-fasted
conscious dogs by use of a dual isotopic method and arteriovenous
difference techniques.
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METHODS |
Animals and surgical procedures.
Sixteen mongrel dogs of either gender (mean weight 23 ± 2 kg) were
studied. Animals were housed in a facility that met American Association for the Accreditation of Laboratory Animals guidelines and
were fed a standard diet of meat and chow (34% protein, 14.5% fat,
46% carbohydrate, and 5.5% fiber based on dry weight). Experimental protocols were approved by the Vanderbilt University School of Medicine
Animal Care Subcommittee. At least 16 days before each experiment, a
laparotomy was performed under general anesthesia. Silastic catheters
(0.03 ID) were inserted in the vena cava for tracer and indocyanine
green (ICG) infusion. A Silastic catheter (0.08 ID) was inserted
through the duodenal wall 3-4 cm below the pylorus for infusion of
cold glucose and
[U-14C]glucose.
Silastic catheters (0.04 ID) were inserted in the portal vein and left
common hepatic vein for blood sampling. Incisions were also made in the
neck region and the inguinal region for the insertion of Silastic
sampling catheters (0.04 ID) in the carotid artery (advanced so that
its tip rested at the aortic arch) and in a lateral circumflex vein
(advanced so that its tip reached the common iliac vein distal to the
anastomosis with the vena cava). After insertion, the catheters were
filled with saline containing heparin, and their free ends were knotted.
Doppler flow probes (Transonic Systems, Ithaca, NY) were used to
measure portal vein, hepatic artery, and external iliac artery blood
flows. A small section of the portal vein upstream from its junction
with the gastroduodenal vein was cleared of tissue, and a 6.0-mm-ID
flow cuff was secured around the vessel. The gastroduodenal vein was
isolated and ligated proximal to its coalescence with the portal vein.
A section of the main hepatic artery proximal to the portal vein was
isolated, and a 3.0-mm-ID flow cuff was placed around the vessel and
secured. The external iliac artery was accessed from the abdominal
incision, dissected free of surrounding tissue, and fitted with a
4.0-mm-ID flow probe cuff that was then secured around the vessel. The
Doppler probe leads and the knotted free catheter ends, with the
exception of the carotid artery and the common iliac vein catheters,
were stored in subcutaneous pockets in the abdominal region so that
complete closure of the skin incision was possible. The carotid artery
and common iliac vein catheters were stored in subcutaneous pockets in
the neck and inguinal regions, respectively.
Only animals that had 1) a leukocyte
count <18,000/mm3,
2) a hematocrit >36%,
3) normal stools, and
4) a good appetite (consuming all of
the daily ration) were used for experiments. Studies were conducted on
conscious dogs after either an 18-h or a 42-h fast. In the dog, an 18-h
fast allows complete gut absorption of a standard meal but induces only
a partial depletion of muscle and liver glycogen stores (7). A 42-h
fast, on the other hand, results in the reduction of muscle and liver
glycogen to a stable minimum, which is <50% of the tissue glycogen
content measured after 18 h of fasting. On the day of the experiment,
the subcutaneous ends of the catheters were freed through small skin
incisions made under local anesthesia (2% lidocaine; Astra
Pharmaceutical, Worcester, MA) in the abdominal, inguinal, and neck
regions. The contents of each catheter were aspirated and were flushed
with saline. Silastic tubing was connected to the exposed catheters and
brought to the back of the dog, where they were secured with
quick-drying glue. Saline was infused through the arterial catheters
throughout the experiment (0.1 ml/min). Data from several of the dogs
in the 18-h-fasted group were included in the database of a previously published study (14).
Experimental procedures.
The study was divided into an equilibration period
(t =
180 to
30 min), a
baseline period (t =
20 to 0 min), and a glucose infusion period (t = 0 to 150 min). At t =
110
min, primers of [3-3H]glucose (30 µCi) and sodium
[14C]bicarbonate (0.64 µCi/min) were given, followed by constant-rate venous infusions of
[3-3H]glucose (0.3 µCi/min) and ICG (0.1 mg/min), which were continued for the duration
of the study. From t = 0 to 150 min,
glucose mixed with
[U-14C]glucose to give
a specific activity (SA) of ~8,700 dpm/mg was given as a primed
infusion (150 mg/kg; 8 mg · kg
1 · min
1)
into the duodenum. Isotopes were obtained from New England Nuclear (Boston, MA), and ICG was purchased from Hynson, Westcott & Dunning (Baltimore, MD). Arterial samples were drawn at 5-min intervals from
t =
20 to 0 min and
at 15-min intervals from t = 0 to 150 min. Portal, hepatic, and common iliac vein samples were drawn at
t =
20,
10, 0, 15, 30, 60, 90, 120, and 150 min. Portal vein, hepatic artery, and external
iliac artery blood flows were recorded continuously from the frequency
shifts of the pulse sound signal emitted from the Doppler flow probes
(15). At the cessation of the experiment, dogs were euthanized with an
overdose of pentobarbital sodium, an abdominal midline incision was
made, and ~2-g biopsies were taken from the liver. An incision was
then made on the medial aspect of the left limb, and ~2-g biopsies
were taken from three hindlimb skeletal muscles (gastrocnemius,
gracilis, and flexor halucis longus). Upon excision, all tissue samples
were immediately frozen with clamps cooled in liquid nitrogen. The time
interval between the pentobarbital injection and the last biopsy was
<5 min.
Processing of blood and tissue samples.
Plasma glucose levels were determined during the experiments by the
glucose oxidase method with a glucose analyzer (Beckman Instruments,
Fullerton, CA). After the completion of the experiment, plasma and
deproteinized blood samples were stored at
70°C until later
analysis. For the determination of plasma glucose radioactivity, 3H and
14C samples were deproteinized
with barium hydroxide and zinc sulfate and placed over Dowex 50W-X8
(Bio-Rad Laboratories, Richmond, CA) and Amberlite (Rohm and Haas,
Philadelphia, PA) resins. Samples were centrifuged, and the supernatant
was evaporated and reconstituted in 1 ml of water and 10 ml of
Ecolite+ (ICN Biomedicals, Irvine,
CA). Radioactivity was then determined by liquid scintillation counting
with a Beckman LS 5000TD. Whole blood [samples deproteinized by
1:3 dilution in 4% perchloric acid (PCA)] lactate, glycerol,
alanine, glucose, and plasma free fatty acids (FFA) were measured by
enzymatic methods (19) on a Technicon Autoanalyzer (Tarrytown, NY) or
on a Monarch 2000 centrifugal analyzer (Instrumentation Laboratories,
Lexington, MA). Whole blood lactate and glucose radioactivities were
determined on a separate set of deproteinized samples (diluted 1:1 in
8% PCA) according to Okajima et al. (26). The content of
14CO2
in whole blood was determined as previously described (11). Liver and
muscle glycogen mass and radioactivity were measured by a previously
described method (3).
Immunoreactive insulin was measured using a double-antibody system
[interassay coefficient of variation (CV) of 10%] (25). Immunoreactive glucagon was measured in plasma samples containing 50 µl of 500 kallikrein international units/ml of Trasylol (FBA Pharmaceuticals, NYC, NY) by use of a double-antibody system (CV of
7%) modified from the method developed by Morgan and Lazarow (25) for
insulin. Insulin and glucagon antisera, standard glucagon and the
125I-labeled glucagon, and
standard insulin and 125I-labeled
insulin were obtained from Linco Research (St. Charles, MO).
Calculations.
The tracer-determined total rate of glucose appearance
(Ra) was determined by
steady-state equations for isotope
[3-3H]glucose dilution
with a pool fraction of 0.65 (5, 8). The systemic
Ra of the intraduodenal glucose
load was determined by dividing the hepatic
[14C]glucose
production by the glucose SA of the intraduodenal infusate. The
Ra of
[14C]glucose was
determined using non-steady-state equations for isotope
[3-3H]glucose dilution
with a pool fraction of 0.65 (5, 8) by using as SA the ratio of
[3H]glucose to
[14C]glucose. The
systemic Ra of intraduodenal
glucose calculated in this manner will be overestimated by an amount
that is dependent on the rate that
[14C]glucose is
recycled. Endogenous glucose Ra
was calculated by subtracting the systemic
Ra of the intraduodenal glucose
from the total glucose Ra.
Net hepatic balances of lactate, glucose,
14CO2,
alanine, FFA, and glycerol were determined by the formula HAF × ([H]
[A]) + PVF × ([H]
[P]), where [A],
[P], and [H] are the arterial, portal vein, and
hepatic vein substrate concentrations, and HAF and PVF are the hepatic
artery and portal vein blood flows (with the exception of FFA, for
which plasma concentrations and flows were used). With this formula,
net substrate output appears as a positive number and net uptake as a
negative number, unless indicated differently. The load of a substrate
reaching the liver was calculated as [P] × PVF + [A] × HAF. Net hepatic fractional substrate
extractions were calculated as the ratio between net hepatic balance
and hepatic load. Net gut balances were calculated as PVF × ([P]
[A]), and net splanchnic balances
were calculated as (HAF + PVF) × ([H]
[A]).
Net limb balances were calculated as LF × ([A]
[I]). LF is limb blood flow through the external
iliac artery, and [I] is the substrate level in the common
iliac vein. Net limb fractional substrate extraction was calculated as
the net limb substrate uptake divided by the limb substrate load, which
was equal to LF × [A]. The mean ratio of blood to
plasma glucose was calculated for the basal period and the glucose
infusion period for each of the four sampling sites. Plasma glucose
values were then multiplied by their corresponding ratio (i.e., blood
glucose/plasma glucose) to convert them to blood glucose
concentrations. The advantage of using plasma glucose measurements is
that a large number of replicates can be obtained, reducing the
measurement CV. The conversion to blood values alleviates the need for
assumptions regarding the equilibration of substrates between red cell
and plasma water.
Hepatic conversion of glucose to
CO2 was calculated as the net
hepatic
14CO2
production rate divided by the hepatic
[14C]glucose precursor
SA. The precursor SA was considered to be the
[14C]glucose SA in the
inflowing blood to the liver and was calculated as (portal vein
[14C]glucose SA × PVF + artery
[14C]glucose SA × HAF)/(HAF + PVF). Because during a
[14C]glucose infusion
[14C]lactate
accumulates, it is necessary to consider lactate SA when net liver
[14C]lactate uptake is
present. In these experiments, lactate was consistently produced by the
liver and therefore was not included in the calculation of hepatic
glucose metabolism. Assumptions involved in these calculations have
been described in detail previously (28, 30).
Net deposition of glycogen deriving from circulating glucose was
calculated in liver and muscle during the intraduodenal glucose infusion as the fraction of glycogen derived from circulating glucose
([14C]glycogen SA/mean
[14C]glucose precursor
SA during intraduodenal glucose infusion) multiplied by the tissue
glycogen content. It should be noted that, in the liver, this
calculation does not include net deposition of glycogen derived from
the indirect pathway. Cold and radioactive liver
glycogen concentrations were the means of biopsy measurements from the
left lateral and left central lobes. Cold and radioactive muscle
glycogen concentrations were the means of measurements from the
gastrocnemius, gracilis, and flexor halucis longus biopsies.
Statistics were performed using SuperAnova (Abacus Concepts, Berkeley,
CA) on a Macintosh PowerPC. Statistical comparison between groups and
over time were made using ANOVA designed to account for repeated
measures. Specific time points were examined for significance by using
contrasts solved by univariate repeated measures. Statistics are
reported in the corresponding table or figure legend for each variable.
Differences were considered significant when
P < 0.05. Data are expressed as
means ± SE.
 |
RESULTS |
Pancreatic hormone levels.
Arterial plasma insulin levels were similar in the two groups during
the baseline period and rose similarly during the intraduodenal glucose
infusion (Fig.
1A).
Arterial plasma glucagon levels were similar between groups at baseline
and were not significantly affected by glucose infusion in either group
(Fig. 1B).

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Fig. 1.
Arterial plasma insulin and glucagon in 18- and 42-h-fasted dogs
(n = 8 for each group) at baseline and
during an intraduodenal glucose infusion (8 mg · kg 1 · min 1).
Values are means ± SE.
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Blood flows.
Portal vein, hepatic artery, and external iliac artery blood flows
(Table 1) were similar in 42- and
18-h-fasted dogs throughout the study.
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Table 1.
External iliac artery, hepatic artery, and portal vein blood
flows in dogs at baseline and during glucose infusion
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Plasma levels, rates of appearance, and net gut output of glucose.
Basal blood glucose concentrations (Fig. 2)
were similar between groups. During intraduodenal glucose infusion,
glucose concentrations were consistently higher in all vessels in the
42- compared with 18-h-fasted animals
(P < 0.05-0.001 throughout the
glucose infusion period). The arterial-portal vein glucose gradient
(Table 2), positive in both groups in the
basal period, was markedly negative during intraduodenal glucose
infusion. Gradient values were slightly more negative in the
42-h-fasted animals, although a significant difference between groups
was detected only at t = 150 min
(P < 0.05).

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Fig. 2.
Plasma concentrations of glucose in artery, portal vein, hepatic vein,
and common iliac vein of 18- and 42-h-fasted dogs
(n = 8 for each group) at baseline and
during an intraduodenal glucose infusion (8 mg · kg 1 · min 1).
Values are means ± SE. * Significantly different from
18-h-fasted dogs.
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The total systemic Ra of glucose
(Table 3) was not significantly different
between groups at baseline or during glucose infusion. Endogenous
glucose production (Table 3) was similar between groups at baseline and
was similarly suppressed during intraduodenal glucose infusion. The
systemic Ra of intraduodenal
glucose, expressed as area under the time-course curve of this
variable, was significantly greater (P < 0.01) in 42-h-fasted dogs compared with 18-h-fasted dogs (Fig.
3A). Net
gut glucose output, again expressed as area under the time-course
curve, tended (P = 0.065) to be
greater in the animals that underwent the longer fast (Fig.
3B).
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Table 3.
Systemic rate of glucose appearance and endogenous glucose production
in dogs at baseline and during glucose infusion
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Fig. 3.
Systemic rate of appearance of intraduodenal glucose and net gut
glucose output in 18- and 42-h-fasted dogs
(n = 8 for each group) at baseline and
during an intraduodenal glucose infusion (8 mg · kg 1 · min 1).
Values are means ± SE. AUC, area under curve.
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Hepatic glucose metabolism.
Basal net hepatic glucose output was ~1.8
mg · kg
1 · min
1
in both groups. During the intraduodenal glucose infusion, both groups shifted to net hepatic glucose uptake (NHGU), with values progressively increasing for the first 60 min of glucose infusion, achieving a
plateau during the final 90 min. There was no difference between 18- and 42-h-fasted animals (Fig.
4C). Net
hepatic glucose fractional extraction was also similar between the two
groups throughout the glucose infusion period (Fig.
4B). The hepatic glucose load was
similar between groups in the basal period but was significantly higher
in the 42-h-fasted animals during the intraduodenal glucose infusion
(P < 0.05-0.001 at
t = 30, 60, 90, and 150 min). Despite similar rates of net hepatic uptake, the intrahepatic fate of the
glucose taken up by the liver was different in the two groups of dogs.
The rate of glucose oxidation was ~35% lower in the 42-h-fasted than
in the 18-h-fasted dogs (Fig. 5).
Conversely, the rate of net liver glycogen synthesis was significantly
higher in the 42-h-fasted than in the 18-h-fasted animals (Fig. 5).
Despite the higher glycogen synthetic rate, the liver glycogen content
was still lower at the end of the intraduodenal glucose infusion in the
42-h-fasted compared with the 18-h-fasted dogs [27.2 ± 5.8 vs.
38.1 ± 6.4 mg/g liver tissue; not significant (NS)] because of
the lower initial glycogen concentration in the 42-h-fasted dogs.

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Fig. 4.
Hepatic glucose load, net hepatic glucose fractional extraction, and
net hepatic glucose balance in 18- and 42-h-fasted dogs
(n = 8 for each group) at baseline and
during an intraduodenal glucose infusion (8 mg · kg 1 · min 1).
Values are means ± SE. * Significantly different from
18-h-fasted dogs.
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Fig. 5.
Mean rates of net liver glycogen synthesis and liver glucose oxidation
in 18- and 42-h-fasted dogs (n = 8 for
each group) during 150 min of intraduodenal glucose infusion (8 mg · kg 1 · min 1).
Liver glucose oxidation data refer to 60-150 min of the glucose
infusion period. Values are means ± SE.
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Hindlimb glucose uptake and fractional extraction, muscle glycogen
content, and net muscle glycogen synthetic rate.
Basal net hindlimb glucose uptake was similar between groups (Table
4). During intraduodenal glucose infusion,
net hindlimb glucose uptake increased similarly in the two groups. Net
hindlimb glucose fractional extraction (Table 4) was also similar in
the two groups throughout the study. The mean glycogen content in skeletal muscle at the end of the experiment was 6.2 ± 0.8 mg/g muscle tissue in the 18-h-fasted dogs and 5.2 ± 0.5 mg/g in
42-h-fasted animals (NS). The net muscle glycogen synthetic rate was
3.8 ± 0.9 mg · g
1 · min
1
in the 18-h-fasted group and 4.5 ± 1.2 mg · g
1 · min
1
in the 42-h-fasted group (NS).
Blood levels and hepatic and hindlimb balances of lactate.
Basal lactate concentrations (Fig. 6) were
significantly lower in the 42-h-fasted dogs compared with 18-h-fasted
dogs in arterial, portal vein, hepatic vein, and common iliac vein
blood. Although both groups displayed a similar pattern of change in
circulating lactate levels during intraduodenal glucose, lactate
concentrations remained significantly lower in the 42-h-fasted animals
throughout the study.

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Fig. 6.
Blood concentrations of lactate in the artery, portal vein, hepatic
vein, and common iliac vein of 18- and 42-h-fasted dogs
(n = 8 for each group) at baseline and
during an intraduodenal glucose infusion (8 mg · kg 1 · min 1).
Values are means ± SE. * Significantly different from
42-h-fasted dogs.
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At baseline, 18-h-fasted dogs displayed net hepatic lactate output
(~2
µmol · kg
1 · min
1),
whereas 42-h-fasted animals displayed a net uptake of ~8
µmol · kg
1 · min
1
(Fig.
7A).
During intraduodenal glucose, 18-h-fasted dogs increased their net
hepatic lactate output to ~15
µmol · kg
1 · min
1.
Although 42-h-fasted animals also became net producers of lactate during intraduodenal glucose, the rates were much lower than in 18-h-fasted dogs (~3
µmol · kg
1 · min
1).

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Fig. 7.
Net hepatic and hindlimb lactate balances in 18- and 42-h-fasted dogs
(n = 8 for each group) at baseline and
during an intraduodenal glucose infusion (8 mg · kg 1 · min 1).
Values are means ± SE. * Significant difference
between 18-and 42-h-fasted dogs.
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During the basal period, 18-h-fasted dogs displayed a net hindlimb
lactate uptake of ~4
µmol · kg
1 · min
1,
which increased to >30
µmol · kg
1 · min
1
during intraduodenal glucose (Fig.
7B). Conversely, 42-h-fasted animals
had a basal net hindlimb lactate output of ~20
µmol · kg
1 · min
1,
which was reduced by intraduodenal glucose but never shifted to net uptake.
Arterial levels, net hepatic uptake and fractional extraction, and
net hindlimb output of alanine.
Basal arterial alanine (Table 5) was lower
in 42- than in 18-h-fasted dogs. The level increased in 42-h-fasted
dogs but was unchanged in 18-h-fasted dogs in response to the glucose
load. Net hepatic alanine uptake (Table 5), although virtually the same
in the two groups of dogs, increased significantly in the 42-h-fasted
animals as a result of the increase in alanine concentration.
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Table 5.
Arterial concentration, net hepatic uptake and fractional extraction,
and net hindlimb uptake of alanine in dogs at baseline and during
glucose infusion
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Arterial levels and net hepatic uptakes and fractional extractions
of glycerol and FFA.
Arterial glycerol (Table 6) was
consistently higher in 42- than in 18-h-fasted dogs. Net hepatic
glycerol uptake was moderately elevated in the 42-h-fasted dogs
compared with 18-h-fasted dogs (P < 0.05 at t = 60 and 120 min) despite a
reduced net hepatic fractional glycerol extraction
(P < 0.05 at
t = 15, 30, 90, and 150 min). Arterial
FFA concentrations were similar in 42- and 18-h-fasted dogs except at
t = 15 min, when it was reduced in 42-h-fasted dogs (Table 6). Net hepatic FFA uptake and fractional extraction, lower in the 42-h-fasted dogs at baseline, were similar between the two groups during intraduodenal glucose.
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Table 6.
Glycerol and FFA arterial concentration, net hepatic balance and net
hepatic fractional extraction in dogs at baseline and during
glucose infusion
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DISCUSSION |
The results of the present study show how a prolonged fast (42 h)
affects the metabolic response to an intraduodenal glucose load. There
was a 60% greater increase in the blood glucose response to
intraduodenal glucose in 42-h-fasted dogs than in the animals fasted
for 18 h. Although the greatest difference in blood glucose concentrations was measured in the portal vein, blood glucose was also
consistently higher in the artery and hepatic vein of the animals that
underwent a longer fast. The onset of the glycemic response was very
rapid. In both 18- and 42-h-fasted dogs, ~75% of the increase in
circulating glucose occurred in the first 15 min after the start of the
intraduodenal glucose infusion; the remaining 25% occurred in the
ensuing 15 min. Subsequent changes in glucose were similar in 18- and
42-h-fasted dogs, but at this time the difference in circulating levels
between the two groups had already been established.
The cause of the greater blood glucose response observed in the dogs
that underwent a longer fast was not a difference in net glucose uptake
by the liver or by the skeletal muscle, as similar rates of these
variables were measured in both groups of animals. However, because
insulin levels were similar and glucose levels were higher after a more
prolonged fast, it is possible that the longer fast duration induced
some degree of resistance to insulin or hyperglycemia. This resistance
may therefore have been one of the determinants of the greater blood
glucose response after prolonged fasting. Part of the greater blood
glucose response after the prolonged fast can be accounted for by
greater intestinal absorption. This is suggested by the fact that the
systemic appearance of intraduodenal glucose, as determined by tracing
the rate of appearance of
[14C]glucose mixed
into the intraduodenal glucose infusate (area under time-course curve,
P < 0.01), was higher in 42- than in 18-h-fasted dogs. Net gut glucose output, as determined by direct measurement of arteriovenous differences, also tended to be higher in
these animals (area under time-course curve,
P = 0.065). Shortly after the start of
the glucose infusion, when the greater blood glucose response was
established, the absolute values of systemic appearance of
intraduodenal glucose and net gut glucose output were similar within
each group. Toward the end of the study, on the other hand, systemic
appearance of intraduodenal glucose was greater than net gut glucose
output. This discrepancy can probably be explained by the recycling of
labeled [14C]glucose
causing a gradual increase in the systemic rate of appearance of
intraduodenal glucose (0-120 min). Because the gradual increase was similar in the 18- and 42-h-fasted groups, the
absolute rates rose equally regardless of the fast
duration. Net gut glucose output, on the other hand, remained constant
in both groups after the initial increase after the onset of the
glucose infusion. The kinetic behavior of this variable, therefore,
paralleled the time course of the circulating glucose levels, although
the difference in net gut glucose output was of a smaller magnitude
(~10%). Because, as described below, uptake of glucose occurs in the
gut during intraduodenal glucose infusion, net gut glucose output
underestimates intestinal glucose absorption.
The rate of net gut glucose output measured in the 42-h-fasted dogs,
when we consider that net gut glucose uptake is ~0.8 mg · kg
1 · min
1
under hyperglycemic conditions (22), shows that ~65% of the infused
sugar was absorbed as glucose. This is similar to the finding of Moore
el al. (24). These investigators showed that, of the total mass of
glucose infused over 4 h, ~27% was not absorbed as glucose. Of this
fraction, a small amount (<5%) could be accounted for by the net gut
output of lactate, alanine, and glycerol. The authors speculated that
the rest could be accounted for by gastrointestinal glucose oxidation
or by glucose that remained in the gut at the end of the study.
The potential mechanisms that might modulate intestinal glucose
absorption after a fast, such as exercise or other physiological stimuli, remain to be determined. Prior exercise is unable to induce a
significant increase in the absorption of water and solutes unless
carbohydrates are part of the ingested solution (13). It is therefore
unlikely that a nonspecific increase in gut permeability might explain
the postexercise increase in carbohydrate absorption. The presence of
glucose in the gut stimulates its own transport into the enterocytes.
This involves Na-dependent glucose transporters on the brush-border
membrane (sodium-dependent D-glucose transporter 1 and 2) and the
Na-independent transporter GLUT-5 (29). The density and activity of
these transporters may be up- or downregulated by
transcriptional/translational processes, dietary manipulation (particularly absence or excess of dietary lipids), and pathological states such as diabetes and inflammatory bowel disease (28). Although
the regulatory signals responsible for transport induction across the
enterocytic membrane are not clearly understood, several gut regulatory
peptides, such as epidermal growth factor and peptide YY, have been
proposed to have important roles in the control of this process (2).
Among the known determinants of NHGU, the arterial and portal vein
insulin and glucagon levels, as well as the arterial-portal vein
glucose gradient, were similar between the two groups. Only the hepatic
glucose load was higher (~15%) in 42-h-fasted dogs. This did not,
however, provide a sufficient stimulus to enhance NHGU detectably in
42-h-fasted dogs beyond rates in the 18-h-fasted dogs. Virtually all
the glucose taken up by the liver in these studies could be accounted
for by oxidation, incorporation into liver glycogen, or release as
lactate. The total amount of glucose that was disposed of in the liver
by the summation of the above processes was the same in 42- and
18-h-fasted dogs (2.3 mg · kg
1 · min
1
in either group). When each pathway is analyzed separately, however, marked differences between groups can be seen. The mean rate of net
glycogen synthesis was over threefold greater in 42- than in
18-h-fasted dogs (1.8 vs. 0.5 mg · kg
1 · min
1).
Conversely, 18-h-fasted dogs displayed a sevenfold greater net lactate
output (1.3 vs. 0.2 mg · kg
1 · min
1)
and a 60% greater rate of liver glucose oxidation (0.5 vs. 0.3 mg · kg
1 · min
1)
than 42-h-fasted animals. These data show how prior fast duration can
markedly alter the intrahepatic flow of carbons. Also, our data suggest
that the suppression of net hepatic lactate production is a mechanism
that determines the blunting of hyperlactatemia in response to glucose
administration after prolonged fasting (10). Changes in the
intracellular levels of some key intermediates in the glycolytic
pathway, such as glucose-6-phosphate, may be the basis for the
differences in the intrahepatic fate of glucose that occur with fasting.
The basal liver glycogen content of our experimental animals can be
estimated by subtracting the net hepatic glycogen synthesis, measured
during glucose infusion, from the final glycogen content. In
18-h-fasted dogs, the mean rate of net liver glycogen synthesis was
~0.5
mg · kg
1 · min
1,
which corresponds to ~2.5 mg of net glycogen storage per gram of
liver tissue over the 150 min of glucose infusion. Subtraction of this
amount from the final glycogen content of 38 ± 5 mg/g yields an
estimated initial glycogen content of ~35.5 mg/g. In 42-h-fasted
dogs, the mean rate of net liver glycogen synthesis was ~1.8
mg · kg
1 · min
1,
which corresponds to ~9 mg of net glycogen storage per gram of liver
tissue over the 150 min of glucose infusion. Subtraction of this amount
from the final glycogen content of 27 ± 6 mg/g yields an estimated
initial glycogen content of ~18 mg/g liver, or one-half the baseline
glycogen estimated to be present in the livers of 18-h-fasted animals.
These calculated values of basal liver glycogen content are similar to
those reported by other investigators for similar fast durations in the
dog (16, 24). It should be noted that the above calculation considers
only glycogen deposition via the direct pathway and therefore
overestimates the initial glycogen content by the amount of the
incorporation of amino acids and glycerol into glycogen. Although the
net hepatic uptakes of alanine and glycerol were measured, it is
impossible to estimate the percentage that was converted to glucose.
The difference in net uptake of either metabolite, however, was
quantitatively small, and greater uptake, if present, was always
measured in the 42-h-fasted group. This indicates that, although the
absolute value of initial glycogen content may have been overestimated, the estimate of the difference between the two groups was probably reasonable.
Fery et al. (10) previously investigated the response to a 75-g oral
glucose load in 14- or 110-h-fasted humans. The arterial glucose level,
whole body glucose oxidation, and storage responses to oral glucose in
these studies are consistent with our results. These investigators,
however, report a delayed and prolonged intestinal absorption of
ingested glucose after the longer fast. This difference probably
reflects the fast durations studied and the presence of delayed gastric
emptying in the experimental model used by these authors
(10). In the 110-h-fasted humans, marked changes in basal
arterial glucagon (>100% increase), insulin (~60% decrease), and
glucose (~45% decrease) occurred that are not present in the 42-h-fasted dogs of the current study. The results obtained in this
study in the dog may reflect a response that is related to the
depletion of glycogen per se, independent of changes in basal circulating glucose and pancreatic hormones.
Despite the fact that circulating glucose levels were consistently
higher in 42- compared with 18-h-fasted animals, arterial insulin
levels were similar between the two groups throughout the study.
Findings similar to those of the 42-h-fasted dogs were reported by
Hamilton et al. (14) in the postexercise state, and others have also
shown that glucose-stimulated insulin secretion is attenuated by prior
exercise in rats and humans (17, 23). The findings of the present study
suggest that this blunting effect on insulin secretion may not be a
specific postexercise effect but may result from the degree of glycogen
depletion or some other adaptation that is common to fasting and exercise.
Arterial glycerol was higher in 42-h-fasted dogs compared with
18-h-fasted dogs, whereas FFA concentrations were similar between the
two groups. This is consistent with observations for similar fast
durations (12, 14) and may reflect a blunting of the antilipolytic
effect of insulin associated with a higher rate of FFA reesterification
after a longer fast (4). Consistent with the difference in arterial
glycerol levels between 18- and 42-h-fasted dogs, a longer fast
increased net hepatic glycerol uptake but did not affect the net
hepatic balance of FFA of the circulating FFA levels.
In summary, prolongation of fast duration before an intraduodenal
glucose load resulted in elevated circulating glucose levels and, to a
smaller extent, increased glucose absorption from the gut. A prolonged
fast did not change NHGU but was associated with increased hepatic
storage of glucose as glycogen, as well as with reduced hepatic glucose
oxidation and net lactate output. These effects occurred although
circulating concentrations of pancreatic hormones were unaffected by
the prior fast duration. The metabolic responses of gut and liver were
similar to those observed after prior exercise. Net muscle glucose
uptake and glycogen synthesis, on the other hand, were not altered by
extending the fast duration from 18 to 42 h, as they are by prior
exercise. Our data emphasize the role played by the interaction of
splanchnic glucose metabolism and nutritional status in the
determination of oral glucose tolerance.
 |
ACKNOWLEDGEMENTS |
We thankfully acknowledge Wanda Snead, Pam Venson, and Brittina
Murphy for excellent technical assistance.
 |
FOOTNOTES |
This work was supported by the National Institute of Diabetes and
Digestive and Kidney Diseases Grant DK-50277. P. Galassetti was
supported by the National Institutes of Health Training Grant DK-07061.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: P. Galassetti, 702 Light Hall, Vanderbilt
Univ. Medical Center, 22nd and Garland Sts., Nashville, TN
37232-0615.
Received 9 June 1998; accepted in final form 19 November 1998.
 |
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