Prior exercise and the response to insulin-induced
hypoglycemia in the dog
Yoshiharu
Koyama1,
Pietro
Galassetti1,
Robert H.
Coker1,
R. Richard
Pencek1,
D. Brooks
Lacy2,
Stephen N.
Davis1,2, and
David H.
Wasserman1,2
1 Department of Molecular Physiology and Biophysics
and 2 Diabetes Research and Training Center,
Vanderbilt University School of Medicine, Nashville, Tennessee
37232-0615
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ABSTRACT |
To test whether hepatic
insulin action and the response to an insulin-induced decrement in
blood glucose are enhanced in the immediate postexercise state as they
are during exercise, dogs had sampling (artery, portal vein, and
hepatic vein) catheters and flow probes (portal vein and hepatic
artery) implanted 16 days before a study. After 150 min of moderate
treadmill exercise or rest, dogs were studied during a 150-min
hyperinsulinemic (1 mU · kg
1 · min
1) euglycemic
(n = 5 exercised and n = 9 sedentary)
or hypoglycemic (65 mg/dl; n = 8 exercised and
n = 9 sedentary) clamp. Net hepatic glucose output
(NHGO) and endogenous glucose appearance (Ra) and utilization (Rd) were assessed with arteriovenous and
isotopic ([3-3H]glucose) methods. Results show that,
immediately after prolonged, moderate exercise, in relation to
sedentary controls: 1) the glucose infusion rate required to
maintain euglycemia, but not hypoglycemia, was higher; 2)
Rd was greater under euglycemic, but not hypoglycemic conditions; 3) NHGO, but not Ra, was suppressed
more by a hyperinsulinemic euglycemic clamp, suggesting that hepatic
glucose uptake was increased; 4) a decrement in glucose
completely reversed the enhanced suppression of NHGO by insulin that
followed exercise; and 5) arterial glucagon and cortisol
were transiently higher in the presence of a decrement in glucose. In
summary, an increase in insulin action that was readily evident under
euglycemic conditions after exercise was abolished by moderate
hypoglycemia. The means by which the glucoregulatory system is able to
overcome the increase in insulin action during moderate hypoglycemia is
related not to an increase in Ra but to a reduction in
insulin-stimulated Rd. The primary site of this reduction
is the liver.
liver; metabolism; oxidation; glucose; lactate; nonesterified fatty
acids
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INTRODUCTION |
IT IS WELL RECOGNIZED
THAT the ability of insulin to stimulate glucose uptake is
enhanced by a single bout of exercise (21). We have shown
that the marked increase in insulin action during exercise is matched
by an equally profound increase in insulin-induced hypoglycemic
counterregulation in an exercising dog model (28). The
effectiveness of this potent counterregulatory response may be
important in decreasing the magnitude and frequency of hypoglycemia during a bout of exercise. Skeletal muscle remains more sensitive to
insulin for a sustained period after exercise (21). In
contrast, the counterregulatory response to insulin-induced
hypoglycemia is actually reduced a full day after prolonged exercise
(9, 16). This is significant, because this likely
contributes to the high risk of hypoglycemia in people with
insulin-dependent diabetes (27). Whether hypoglycemic
counterregulation during the interval immediately after exercise
remains sensitized, as it is during exercise, or blunted, as it is
after extended recovery from exercise, is unknown.
These studies test whether sensitization of the counterregulatory
response to insulin-induced hypoglycemia is present during the early
stages of recovery from prolonged exercise (<4 h after exercise) and
the tissue-specific metabolic consequences of any sensitization.
Experiments were conducted after 150 min of exercise or an
equivalent-duration sedentary period in chronically catheterized, overnight-fasted dogs. Glucose fluxes and the tissue-specific fates of
glucose in liver and muscle were assessed using isotopic ([3-3H]glucose, [U-14C]glucose) and
arteriovenous difference (liver, limb) techniques.
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METHODS |
Animal maintenance and surgical procedures.
Mongrel dogs (n = 32, mean wt 22.9 ± 0.3 kg) of
either gender that had been fed a standard diet (Pedigree, Vernon, CA)
and Wayne Lab Blox (51% carbohydrate, 31% protein, 11% fat, and 7% fiber based on dry weight; Allied Mills, Chicago, IL) were studied. The
dogs were housed in a facility that met American Association for the
Accreditation of Laboratory Animal Care guidelines, and the Vanderbilt
University School of Medicine Animal Care Committee approved the
protocols. At least 16 days before each experiment, a laparotomy was
performed with animals under general anesthesia (25 mg/kg pentobarbital
sodium). Silastic catheters (0.03 in. ID) were inserted into the vena
cava for tracer ([3-3H]glucose,
[U-14C]glucose), indocyanine green (ICG), and insulin
infusions. Silastic catheters (0.04 in. ID) were also inserted into the
portal vein and left common hepatic vein for blood sampling. Incisions
were made in the neck and inguinal regions for the placement of carotid artery and common iliac vein sampling catheters, respectively. The
carotid artery was isolated, and a Silastic catheter (0.04 in. ID) was
inserted and advanced so that its tip rested in the aortic arch. A
Silastic catheter (0.03 in. ID) was introduced into the common iliac
vein via a lateral circumflex vein. Exposure of the lateral circumflex
vein was achieved with a 2-cm incision in the lower femoral region and
dissection of the vein from the subcutaneous tissues. The catheter tip
was positioned in the common iliac vein, distal to the anastomosis with
the vena cava. The median sacral vein was ligated to prevent dilution
from other sites. Verification of catheter placement was made through
the abdominal incision site. After insertion, all catheters were filled with saline containing heparin (200 U/ml; Abbott Laboratories, North
Chicago, IL), and their free ends were knotted.
A Doppler flow probe was used to measure external iliac artery blood
flow (11). Briefly, 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, which was then secured around
the vessels. Catheters and the Doppler probe lead were stored under the
skin at the site where access to vessels was achieved (i.e., abdominal,
neck, and inguinal regions).
Starting 1 wk after surgery, dogs were accustomed to running on a
motorized treadmill regardless of whether they were used for sedentary
or exercise experiments. Animals were not exercised during the 48 h preceding an experiment. Only animals that had 1) a
leukocyte count <18,000/mm3, 2) a hematocrit
>36%, 3) normal stools, and 4) a good appetite (consuming the entire daily ration) were used.
Studies were conducted after an 18-h fast, because dogs are
postabsorptive after this interval. On the day of the experiment, the
subcutaneous ends of the catheters were freed from subcutaneous pockets
through small skin incisions made under local anesthesia (2%
lidocaine; Astra Pharmaceutical Products, Worcester, MA). The contents
of each catheter were aspirated, and the catheters were flushed with
saline. Silastic tubing was connected to the exposed catheters and
secured to the back of the dog using quick-drying glue. Saline was
infused in the arterial catheter throughout the experiments (0.1 ml/min).
Experimental procedures.
Protocols are illustrated in Fig. 1.
Experiments consisted of a period of moderate-intensity (100 m/min,
12% grade) treadmill exercise or rest (
200 to
50 min), a basal
sampling period (
40 to 0 min), and a euglycemic or hypoglycemic clamp
period (0 to 270 min). Primed, constant-rate infusions of
[3-3H]glucose (primer of 42 µCi, infusion of 0.30 µCi/min) and [U-14C]glucose (primer of 24 µCi,
infusion of 0.17 µCi/min) were initiated at
120 min and continued
throughout the study. In addition, a bolus of sodium
[14C]bicarbonate (0.64 µCi/kg) was given, and an
infusion of ICG (0.1 mg · m
2 · min
1) was
initiated at this time. At t = 0 min, an infusion of
insulin (1 mU · kg
1 · min
1)
was started. Arterial glucose was clamped at euglycemic levels in nine
sedentary and six exercising dogs and at moderately hypoglycemic levels
in nine sedentary dogs and eight exercising dogs. During euglycemic
clamps, [3-3H]glucose was mixed with the cold glucose
infusate to minimize changes in [3-3H]glucose specific
activity. During hypoglycemic clamps, the [3-3H]glucose
was adjusted, with the goal again of maintaining
[3-3H]glucose specific activity constant. Arterial
samples were drawn at 10-min intervals from t =
40 to
150 min. Portal, hepatic, and common iliac vein samples were drawn at
t =
40,
20, 0, 10, 30, 50, 60, 80, 100, 110, 130, and 150 min. External iliac artery blood flow was recorded continuously
from the frequency shifts of the pulsed sound signal emitted from the
Doppler flow.

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Fig. 1.
Protocols contained a dye and isotope equilibration period ( 120
to 40 min), a period of moderate-intensity treadmill exercise or rest
( 200 to 50 min), and a basal sampling period ( 40 to 0 min).
Insulin was infused from t = 0 to 150 min (1 mU · kg 1 · min 1), and
arterial plasma glucose levels were clamped at euglycemic (~110
mg/dl) or moderately hypoglycemic (~65 mg/dl) levels. There were
(n) 9 sedentary animals under euglycemic and hypoglycemic
conditions, 6 animals under the exercised euglycemic condition, and 8 under the exercised hypoglycemic condition.
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Processing of blood samples.
Plasma glucose levels were determined by the glucose oxidase method
with a Beckman glucose analyzer (Beckman Instruments, Fullerton, CA).
Plasma glucose radioactivity (3H and 14C)
samples were deproteinized with barium hydroxide and zinc sulfate and
subsequently placed over Dowex 50W-X8 (Bio-Rad Laboratories, Richmond,
CA) and Amberlite (Rohm and Haas, Philadelphia, PA) resins. Resined
samples were centrifuged, and the supernatant was evaporated and
reconstituted in 1 ml of water and 10 ml of Ecolite+ (ICN, Irvine, CA).
Radioactivity was then determined by dual-label liquid scintillation
counting (model LS-5000TD, Beckman Instruments. Fullerton, CA). ICG
concentration was measured spectrophotometrically (805 nm) in arterial
and hepatic vein plasma immediately after experiments. Plasma
nonesterified fatty acids (NEFA) were measured spectrophotometrically
by use of the kit made by Wako Chemicals (Richmond, VA). Whole blood
lactate, alanine, and glycerol concentrations were determined in
samples deproteinized with an equal volume of 8% perchloric acid by
enzymatic methods (13) on a Monarch 2000 centrifugal
analyzer (Instrumentation Laboratories, Lexington, MA). The
14CO2 in whole blood was liberated by
acidification with hydrochloric acid, trapped on chromatography paper
using hyamine hydroxide, and quantified using liquid scintillation counting.
Immunoreactive insulin was measured using a double-antibody system
[interassay coefficient of variation (CV) of 10%]. Immunoreactive glucagon was measured in plasma samples containing 50 µl of 500 kallikrein inhibitory units/ml aprotinin (Trasylol; FBA
Pharmaceuticals, New York) with a double-antibody system (interassay CV
of 7%) modified from the method developed for insulin. Plasma
norepinephrine and epinephrine levels were determined using a
high-performance liquid chromatography technique (interassay CVs of 11 and 13%, respectively). Plasma cortisol was measured using the
clinical Assays Gamma Coat Radioimmunoassay Kit (Clinical Assays,
Travenol-Genetech Diagnostics, Cambridge, MA; interassay CV of 6%).
The methods used for hormone analyses by this laboratory have been
described previously (2).
Materials.
High-performance liquid chromatography-purified
[3-3H]glucose and [U-14C]glucose (New
England Nuclear, Boston, MA) were used as the glucose tracers. ICG was
purchased from Hynson, Westcott and Dunning (Baltimore, MD). Glucagon
and insulin antisera were obtained from Dr. R. L. Gingerich
(Washingtion University School of Medicine, St. Louis, MO), and the
standard glucagon and 125I-labeled glucagon were obtained
from Novo Research Institute (Copenhagen, Denmark). Standard insulin
and 125I-labeled insulin were obtained from Linco Research
(St. Louis, MO). Enzymes used in chemical analyses were obtained from
Sigma Chemical (St. Louis, MO) or Boehringer-Mannheim Biochemicals
(Mannheim, Germany). Doppler flow probes were obtained from the
Instrumentation Development Laboratory, Baylor University School of Medicine.
Calculations.
Total glucose appearance (Ra) and disappearance
(Rd) were determined using the two-compartment model
described by Mari (14) for nonsteady state with
[3-3H]glucose as the tracer. Endogenous glucose
production (also Ra) was calculated as the total glucose
production minus the exogenous glucose infusion rate. The
two-compartment approach does not result in the underestimate of
Ra values seen under hyperinsulinemic conditions when the
modified one-compartment model is used (14). The accuracy
of the Ra calculation was further strengthened by using a
variable [3-3H]glucose infusion rate during the
experimental period to minimize changes in glucose specific activity.
Net hepatic glucose balance was determined by the formula
HAF · ([A]
[H]) + PVF · ([P]
[H]),
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 (or plasma) flows, respectively. The dye
dilution technique gives a measure of total hepatic blood flow. It was
assumed on the basis of extensive experience in our laboratory that HAF
and PVF are 20 and 80% of total hepatic flow, respectively (2,
10). In the calculations of net hepatic glucose balance, the
sign (+ or
) was reversed so that net output would be a positive number.
Net limb balances were calculated as LF · ([A]
[I]). LF
is limb blood flow through the external iliac artery and is the substrate level in the common iliac vein, and [I] is the iliac vein
substrate concentration. The sign was reversed for the calculation of
limb 14CO2 production. Limb fractional glucose
extraction was calculated as the net limb glucose uptake (LGU) divided
by the limb glucose load (LF · [A]). Blood levels and flows
were used for the calculation of all hepatic and limb balances with the
exception of those for NEFA. Whole blood glucose values were assumed to
be 73% of plasma glucose values on the basis of numerous comparisons
made previously in the Vanderbilt Diabetes Research and Training Center
and in conjunction with the present study. The use of blood values, and not plasma, alleviates the need for assumptions regarding the equilibration of substrates between red cell and plasma water.
Limb glucose oxidation (LGO) was calculated as the limb
14CO2 output divided by the specific activity
of glucose. Limb glucose nonoxidative metabolism (LGNO) was determined
as the difference between net limb glucose uptake and oxidation.
Assumptions involved in these calculations have been described in
detail previously (24).
Statistics were performed using StatView (Abacus Concepts, Berkeley,
CA) and SuperANOVA (Abacus Concepts) on a Macintosh computer. Because
hormones and metabolism change as a function of time after exercise,
the statistical comparisons that are emphasized are those made at the
same interval after exercise (or corresponding sedentary period). This
in effect normalized for the dynamic nature of exercise recovery.
Statistical comparisons between groups and over time were made using
analysis of variance designed to account for repeated measures.
Specific time points were examined for significance by using contrasts
solved by univariate repeated measures. Statistics are shown for each
variable in its corresponding table or figure. Differences were
considered significant when P values were <0.05. Exercise
values presented in Tables 1-6 are the means of three data points
within the designated 40-min intervals. Data are expressed as
means ± SE.
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RESULTS |
Arterial plasma insulin and glucose and glucose infusion rates.
Arterial plasma insulin rose from a basal value of ~6 to ~40
µU/ml during the test period in all protocols (Fig.
2). Arterial glucose levels were clamped
at euglycemic (114 ± 7 mg/dl in sedentary dogs and 107 ± 5 mg/dl in exercise dogs) or moderately hypoglycemic (68 ± 3 mg/dl
in sedentary dogs and 67 ± 2 mg/dl in exercise dogs) levels (Fig.
2).

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Fig. 2.
Arterial plasma glucose and insulin concentrations during
hyperinsulinemic euglycemic and hypoglycemic clamps. Data are
means ± SE; n = 9 animals under sedentary
euglycemic and hypoglycemic conditions, 6 under the exercised
euglycemic condition, and 8 under the exercised hypoglycemic
condition.
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The glucose infusion rates required to clamp glucose at euglycemic
levels were ~10 and ~8
mg · kg
1 · min
1 in
exercised and sedentary dogs (Fig. 3).
This difference of ~2
mg · kg
1 · min
1 was
significant. In relation to the euglycemic clamps, the glucose infusion
rates required for hypoglycemic clamps were markedly reduced. Rates
were
1 mg · kg
1 · min
1 in
both exercised and sedentary dogs. The reduction in glucose infusion
rate required for the hypoglycemic clamp was significantly more in the
postexercise state (Fig. 4), reflecting
an improved ability to counterregulate in the state immediately after
exercise.

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Fig. 3.
Effect of prior exercise on glucose infusion rates during
hyperinsulinemic euglycemic and hypoglycemic clamps. Data are
means ± SE; n = 9 sedentary animals under
euglycemic and hypoglycemic conditions, 6 under the exercised
euglycemic condition, and 8 under the exercised hypoglycemic condition.
*Significant difference vs. corresponding hypoglycemic group
(P < 0.05); significant difference vs. corresponding
sedentary group (P < 0.05).
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Fig. 4.
Effect of prior exercise on the decrement in glucose
infusion rates due to moderate hypoglycemia. #, This
variable was calculated as the difference between ~110 and ~65
mg/dl clamps for each condition. Significant difference vs.
corresponding sedentary group (P < 0.05).
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Arterial plasma glucagon, catecholamine, and cortisol levels.
Arterial plasma glucagon was significantly higher in exercised groups
compared with sedentary groups before the onset of insulin infusion
(Table 1). As anticipated, glucagon was
higher during hypoglycemia than during euglycemia whether or not the
animals were exercised (P < 0.05). The increase due to
hypoglycemia was more in exercised dogs compared with sedentary dogs
during the first 50 min (P < 0.05).
Neither arterial epinephrine nor norepinephrine levels were different
in the postexercise and sedentary states before insulin infusion (Table
1). Epinephrine was unchanged during euglycemic clamps but increased
three- to fourfold in response to hypoglycemia. The increase was
similar regardless of whether animals were sedentary or exercised.
Arterial norepinephrine concentrations were not significantly different
under euglycemic clamp conditions compared with hypoglycemic clamp
conditions regardless of whether animals were sedentary or exercised.
Arterial plasma cortisol concentrations were higher in the postexercise
state compared with sedentary conditions before the insulin infusion
(Table 1). Euglycemic clamps in exercised and sedentary dogs did not
significantly affect cortisol levels. Hypoglycemia increased cortisol
levels in exercised and sedentary dogs. It is noteworthy that cortisol
was increased significantly more during the first 50 min of the
hypoglycemic clamp in exercised compared with sedentary dogs.
Glucose kinetics.
Although NHGO tended to be higher before insulin infusion after
exercise, differences between groups were not significant. As expected,
NHGO was significantly suppressed by insulin in the presence of
euglycemia in both sedentary (2.6 ± 0.4 and 0.4 ± 0.2 mg · kg
1 · min
1 in the
basal state and at t = 150 min, respectively) and
exercised (3.3 ± 0.8 and
0.9 ± 0.5 mg · kg
1 · min
1 in the
basal state and at t = 150 min) groups (Fig.
5). NHGO was actually negative (net
uptake) in five of the six exercised dogs and two of the nine sedentary
dogs during euglycemic clamps. Interestingly, the total suppression of
NHGO was ~twofold more in exercised dogs. As expected, hypoglycemia
resulted in a relative stimulation of NHGO so that the suppressive
effects of insulin on this variable were largely overcome in both
sedentary (2.1 ± 0.5 and 1.5 ± 0.3 mg · kg
1 · min
1 in the
basal state and at t = 150 min) and exercised (3.2 ± 0.5 and 2.3 ± 0.6 mg · kg
1 · min
1 in the
basal state and at t = 150 min) dogs. The stimulation of NHGO due to hypoglycemia (i.e., the difference between NHGO during
hypoglycemic and euglycemic clamps) was greater in the postexercise
state (Fig. 6).

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Fig. 5.
Effect of prior exercise on net hepatic glucose output during
hyperinsulinemic euglycemic and hypoglycemic clamps. Data are
means ± SE; n = 9 sedentary animals under
euglycemic and hypoglycemic conditions, 6 under the exercised
euglycemic condition, and 8 under the exercised hypoglycemic condition.
*Significant difference vs. corresponding euglycemic group
(P < 0.05).
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Fig. 6.
Effect of prior exercise on stimulation of net hepatic
glucose output (NHGO) due to a decrement in glucose. This variable was
calculated as the difference between ~110 and ~65 mg/dl clamps for
each condition. Significant difference vs. corresponding sedentary
group (P < 0.05).
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Ra, like NHGO, also tended to be higher before insulin
infusion in exercised compared with rested dogs. During the interval immediately after exercise, the [3-3H]glucose increases
rapidly because of the abrupt decrease in its clearance by muscle. As a
result, Ra values during this period are particularly
vulnerable to the model assumptions used in the calculations.
Nevertheless, differences between groups before clamps were not
significant. During the clamp period, specific activities were within
±25% of basal specific activities in all protocols. As expected,
Ra was also suppressed by insulin under euglycemic
conditions in both sedentary (2.7 ± 0.3 and 1.1 ± 0.4 mg · kg
1 · min
1 in the
basal state and last 30 min of insulin infusion) and exercised (4.2 ± 0.5 and 2.4 ± 0.6 mg · kg
1 · min
1 in the
basal state and and last 30 min of insulin infusion) dogs (Fig.
7). Hypoglycemia led to a stimulation of
Ra so that the suppressive effects of insulin on this
variable were effectively counterbalanced in both sedentary (2.7 ± 0.3 and 2.6 ± 0.5 mg · kg
1 · min
1 in the
basal state and last 30 min of insulin infusion) and exercised (3.5 ± 0.6 and 3.2 ± 0.3 mg · kg
1 · min
1 in the
basal state and last 30 min of insulin infusion) dogs. In contrast to
the suppression of NHGO, the suppression of Ra was not
significantly greater during the exercise recovery period (Fig.
8).

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Fig. 7.
Effect of prior exercise on endogenous glucose appearance rates
during hyperinsulinemic euglycemic and hypoglycemic clamps. Data are
means ± SE; n = 9 sedentary animals under
euglycemic and hypoglycemic conditions, 6 under the exercised
euglycemic condition, and 8 under the exercised hypoglycemic condition.
*Significant difference vs. corresponding hypoglycemic group
(P < 0.05).
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Fig. 8.
Effect of prior exercise on the increase in endogenous
glucose appearance rates due to a decrement in glucose.
#This variable was calculated as the difference between
~110 and ~65 mg/dl clamps for each condition. Data are means ± SE.
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Rd tended to be higher after exercise before the insulin
infusion, but differences were not significant. The increase in
Rd during the euglycemic clamp was less in sedentary
(2.8 ± 0.3 and 8.0 ± 1.3 mg · kg
1 · min
1 in the basal
state and last 30 min of insulin infusion) compared with exercised
(4.0 ± 0.4 and 12.2 ± 1.0 mg · kg
1 · min
1 in the
basal state and last 30 min of insulin infusion) dogs (Fig.
9). Hypoglycemia reduced Rd
in sedentary (2.9 ± 0.3 and 3.0 ± 0.6 mg · kg
1 · min
1 in the
basal state and at t = 150 min) and exercised (3.4 ± 0.6 and 4.0 ± 0.5 mg · kg
1 · min
1 in the
basal state and at t = 150 min) dogs. The reduction in Rd in the postexercise state was significantly greater than
in the sedentary state (Fig. 10).

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Fig. 9.
Effect of prior exercise on glucose disappearance during
hyperinsulinemic euglycemic and hypoglycemic clamps. Data are
means ± SE; n = 9 sedentary animals under
euglycemic and hypoglycemic conditions, 6 under the exercised
euglycemic condition, and 8 under the exercised hypoglycemic condition.
*Significant difference vs. corresponding euglycemic group
(P < 0.05). Significant difference vs. corresponding
sedentary group (P < 0.05).
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Fig. 10.
Effect of prior exercise on the suppression of glucose
disappearance rate due to a decrement in glucose. #This
variable was calculated as the difference between ~110 and ~65
mg/dl clamps for each condition. Significant difference vs.
corresponding sedentary group (P < 0.05).
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LGU, LGO, and LGNO.
LGU, LGO, and LGNO were not different between groups before the insulin
infusion period (Table 2). LGU was
increased markedly during euglycemic clamps in both sedentary and
exercised dogs (Table 2). This increase was greater in exercised dogs,
and it corresponded to a greater rate of LGNO. LGO was also increased during the euglycemic clamps, but the rate was not influenced by prior
exercise. LGU, LGO, and LGNO were all reduced during hypoglycemic
clamps in relation to euglycemic clamps. Rates of limb glucose uptake
and metabolism during hypoglycemic clamps were not influenced by prior exercise.
Arterial concentrations and net balances of lactate.
Table 3 shows that arterial lactate
levels were not significantly affected by prior exercise or insulin
infusion in the presence of a euglycemic or hypoglycemic clamp. It
should be noted that, with regard to net tissue balances of lactate,
the variability inherent in these measurements is high, and small to
moderate differences may go undetected. Net hepatic lactate uptake was not significantly affected by prior exercise. Glycemia was a
significant factor in determining net hepatic lactate balance. The
liver was a net producer of lactate during euglycemic clamps, whereas
during hypoglycemic clamps, the liver was a net consumer of lactate. Differences were significant during the two latter periods of the
insulin infusion. Net limb lactate output was not significantly affected by prior exercise. Although there was a tendency for greater
net limb output during hypoglycemia compared with euglycemia, differences were insignificant.
Arterial concentrations and net balances of glycerol.
Arterial concentrations, net hepatic uptake, and net limb output of
glycerol were not significantly affected by prior exercise (Table
4). Concentrations were suppressed
significantly by insulin during euglycemic clamps in both sedentary and
exercised dogs. Glycerol concentrations exhibited no change during
hypoglycemia after exercise but rose by ~40% in dogs that had
remained sedentary. Net hepatic glycerol uptake was significantly
reduced during euglycemic clamps regardless of whether dogs were
sedentary or exercised. Hypoglycemia caused a significant increase in
net hepatic glycerol uptake relative to corresponding basal periods and
euglycemic clamp periods in both sedentary and exercised dogs. Net limb
glycerol output was suppressed during euglycemic clamps relative to
corresponding basal periods, but it was unchanged from basal during
hypoglycemic clamps.
Arterial plasma NEFA concentrations.
NEFA concentrations were similar in all groups before the insulin
infusion (Table 5). As expected, insulin
suppressed NEFA concentrations under euglycemic conditions. This
suppression was equal in exercised dogs and dogs that had remained
sedentary. This effect of insulin was reduced under hypoglycemic
conditions. NEFA concentrations were significantly higher during
hypoglycemic clamps compared with corresponding intervals during
euglycemic clamps. Again, differences were not significantly different
between exercised dogs and those that had remained sedentary.
Hepatic and limb blood flows.
Hepatic and limb (external iliac artery) blood flows were unaffected by
prior exercise, hyperinsulinemia, or hypoglycemia (Table
6).
 |
DISCUSSION |
It is well established that prior exercise causes increases in
insulin-stimulated whole body glucose disappearance, muscle glucose
uptake, and muscle nonoxidative glucose metabolism (21, 23). The present studies add two important new elements to our knowledge of the effects of prior exercise on insulin-stimulated metabolism. The first is that prior exercise significantly enhances the
action of insulin on the liver, as it does on muscle, under euglycemic
conditions. Despite the greater suppressive effect of insulin,
prior exercise did not influence NHGO during hypoglycemia. The second
new finding, therefore, is that in the presence of moderate
insulin-induced hypoglycemia, there must be added stimulatory drive to
the liver to overcome the enhanced action of insulin at the liver. It
is probable that many factors determine these responses, including the
actual duration after exercise in which measurements are made. Results
obtained from these experiments may be specific for the ~4-h period
after prolonged, moderate-intensity exercise.
With regard to the first finding, prior exercise increased the
suppression of NHGO by a physiological increment in insulin by
~twofold under euglycemic conditions. This finding was readily detectable despite the inherent variability of the arteriovenous difference method. It is important to recognize that the ability of
insulin to suppress Ra was not enhanced. The lack of an
effect of prior exercise on the ability of insulin to suppress
Ra is consistent with findings in human subjects (18,
23). It is noteworthy that the reduction in Ra
during the euglycemic clamp after exercise appeared to wane during the
latter stage of the 150-min clamp, whereas the reduction in NHGO was
sustained. This suggests that the effect of insulin on hepatic glucose
dynamics may be dependent on the time frame after exercise in which
measurements are made. Earlier estimates of Ra that were
done after exercise in human subjects were difficult to assess because
of weaknesses in the tracer method associated with either possible
isotopic impurities (17) or errors in the model used to
calculate glucose fluxes (14). Using an HPLC-purified
product minimized the possibility of isotope contamination in the
present study. The error associated with the model used to calculate
glucose fluxes was addressed in two ways. First, a two-compartment
model that is less dependent on steady-state conditions was used
(14) and not the modified one-compartment model used
previously (18, 23). Moreover, varying the isotope
infusion rate to minimize changes in specific activity reduced model
dependency of the glucose flux calculations.
The observation that suppression of NHGO, but not Ra, was
greater during hyperinsulinemic, euglycemic clamps after exercise strongly suggests that insulin-induced stimulation of hepatic glucose
uptake was increased. This effect of prior exercise was apparent even
though insulin levels were actually lower, albeit not significantly so,
by ~20%, before and during the insulin infusion. Moreover, this
difference was present even though circulating epinephrine levels
actually remained higher after exercise (Table 1). Several studies have
suggested that the ability of the liver to consume glucose after
exercise is improved (7, 8, 12, 15). It is unknown whether
this is due to an insulin-dependent or an insulin-independent
mechanism. One study demonstrated that prior exercise increased
insulin-induced generation of pyruvate dehydrogenase-stimulating
activity in the rat in the absence of any effect on insulin binding to
the hepatocyte membrane (1). This suggested that exercise
enhances hepatic insulin action at a site distal to the hepatic insulin
receptor. The present study is the first of which we are aware to show
that hepatic insulin action is increased in vivo. The mechanism for the
increase in hepatic insulin action remains to be determined. One of the
key determinants of the postexercise increase in muscle insulin action is depletion of muscle glycogen stores (3, 21). The
exercise protocol used in the present study depletes liver glycogen
stores ~70% (26). It is possible that a reduction in
liver glycogen stores could be a cause of the enhanced ability of
insulin to enhance hepatic glucose uptake under euglycemic conditions.
Although the glucose infusion rate was higher in the postexercise state
during the euglycemic clamp, the glucose infusion rates during the
hypoglycemic state were virtually identical in exercised and sedentary
dogs. This shows that the counterregulatory response during the
postexercise period was sufficiently increased to offset the increase
in insulin action. As mentioned earlier, after exercise, insulin was
~20% lower during the insulin clamp period (the difference was
insignificant) because of reduced preclamp levels. This could
counterbalance the effect of the increase in insulin action after
exercise. This is unlikely to be an important variable in these
studies, however, as increased insulin action was demonstrable during
the euglycemic experiments, even though the same insignificant
difference in insulin levels was present. Clearly, then, there is a
distinct difference between the responses in euglycemic and
hypoglycemic clamps with and without prior exercise. The improved
counterregulatory response effectively nullified the exercise-induced
increase in whole body glucose uptake by actions on both muscle and
liver. Table 3 shows that hypoglycemia reduced muscle glucose uptake
and nonoxidative metabolism during insulin clamps but that it did it
twice as well after exercise. LGO and nonoxidative metabolism were
reduced by ~50 µmol/min during hypoglycemic clamps compared with
euglycemic clamps in sedentary dogs. After exercise, however,
hypoglycemia resulted in an ~100 µmol/min reduction in these
variables. Figure 6 showed that hypoglycemia was twice as effective in
stimulating NHGO after exercise. This observation, combined with the
fact that hypoglycemia was no more effective in stimulating
Ra after exercise, indicates that the exercise-stimulated
increase in hepatic glucose uptake was negated by the counterregulatory
response. Equivalent hypoglycemic responses in Ra in
sedentary and exercised dogs, coupled with equivalent increments in
hepatic gluconeogenic precursor (lactate and glycerol) uptake, support
the premise that prior exercise did not promote the formation of
glucose by the liver. The existence of differences in the gluconeogenic
response to hypoglycemia due to prior exercise cannot be completely
ruled out, however; because not all of the gluconeogenic precursors
were measured, the balance of those we did measure was highly variable,
and we have no index of intracellular gluconeogenic regulation.
Research in human subjects shows that the response to an
insulin-induced hypoglycemia of ~50 mg/dl is blunted a full day after 180 min of moderate exercise (50% maximum oxygen uptake; two bouts of
90 min each) (9). This is consistent with a recent report showing that the counterregulatory response to a stepped hypoglycemic stimulus that achieved a nadir of 45 mg/dl was blunted one day after
120 min of moderate exercise (70% maximum oxygen uptake; two bouts of
60 min each) (16). In this latter study, however, the
blunting was somewhat less marked than in the former study. Duration
after exercise seems to be an important parameter, because when
insulin-induced hypoglycemia was induced beginning 60 min after
moderate exercise (~60% maximum oxygen uptake for 60 min), no
blunting was observed in healthy human subjects (20). The mechanism for this time-dependent blunting of the counterregulatory response to hypoglycemia has been proposed to be due to the increment in cortisol that occurs with sufficiently intense exercise (4, 5). In the present study, in which the hypoglycemic period was
in even closer temporal proximity to exercise, there was a transiently
greater arterial glucagon and cortisol response to hypoglycemia.
Granted, it is likely that the increase in portal vein glucagon was
considerably higher and perhaps more persistent than arterial glucagon
during hypoglycemia in exercised dogs compared with sedentary dogs
(19, 25). Epinephrine was insignificantly higher during
the hypoglycemic period. This trend appears to be related to residual
effects of primary exercise rather than to a difference in the response
to hypoglycemia. One cannot rule out, however, that acute exercise
enhances the sensitivity to adrenergic stimulation (22).
The effects of prior exercise on glucose disappearance and muscle
glucose metabolism during insulin-induced hypoglycemia were clearly
sustained throughout the experimental period. Glucose fluxes and the
overall effectiveness of counterregulation were not assessed in the
most comparable human study in which hypoglycemia was induced beginning
60 min after exercise (20).
Lipolysis was, as expected, suppressed by insulin during the euglycemic
clamps. This was evidenced by the reduction in arterial glycerol and
NEFA concentrations and in net limb glycerol output under these
conditions. The magnitude of these decrements was unaffected by prior
exercise. Hypoglycemia countered the suppressive effects of insulin on
lipolysis in both exercised and sedentary dogs. There was no
significant difference in arterial glycerol, arterial NEFA, and net
limb glycerol output during hypoglycemia in the two groups. Glycerol
rose significantly above the preinsulin infusion value, however, only
in the sedentary hypoglycemic group. The lipolytic response did not
increase above preinsulin infusion levels in the exercised hypoglycemic
group. This observation is consistent with an earlier study showing
that prior exercise blunted the lipolytic response to hypoglycemia
(9). The generally minimal differences in lipolytic
indexes between exercised and sedentary dogs are consistent with the
fact that the catecholamine response to hypoglycemia was similar in the
two groups. Catecholamines are the primary stimulators of the lipolytic
response to hypoglycemia (6).
The exercise-induced increase in insulin action persists well after the
cessation of the work bout (21). The duration and magnitude of this persistence are influenced by factors relating to
nutritional state, fitness, and duration and intensity of the prior
work bout (27). Although the response to insulin-induced hypoglycemia after exercise does not appear to be characterized by the
same long-term persistence as other aspects of insulin action (9,
16), it still may be possible that the response is influenced by
characteristics of the subjects tested and by exercise condition.
In summary, differences between the response to an insulin-induced
decrement in arterial glucose and an equivalent duration period of
hyperinsulinemic euglycemia were used to assess the effectiveness of
defenses against a moderate decrement in arterial glucose shortly after
the cessation of exercise. Results show that, when experiments were
initiated immediately after prolonged, moderate exercise and values
were compared with those of sedentary controls, 1) the
glucose infusion rate required to maintain euglycemia, but not
hypoglycemia, was higher, indicating that postexercise insulin
sensitivity was counterbalanced by a counterregulatory response that
was more effective; 2) Rd was greater under
euglycemic, but not hypoglycemic, conditions; 3) NHGO, but
not Ra, was suppressed more by a hyperinsulinemic
euglycemic clamp, suggesting that hepatic glucose uptake was increased;
4) a decrement in glucose completely reversed the more
potent effects of insulin on NHGO; and 5) arterial glucagon
and cortisol were transiently higher in the presence of a decrement in
glucose. In summary, an increase in insulin action that was readily
evident under euglycemic conditions shortly after the cessation of
exercise was abolished by moderate hypoglycemia. The means by which the
glucoregulatory system is able to overcome the increase in insulin
action during moderate hypoglycemia is related not to an increase in
Ra but to a reduction in insulin-stimulated Rd.
The primary site at which this reduction occurs is the liver.
 |
ACKNOWLEDGEMENTS |
We are grateful to Eric Allen, Wanda Snead, and Angie Penazola for
excellent technical assistance.
 |
FOOTNOTES |
This work was supported by National Institute of Diabetes and Digestive
and Kidney Diseases Grant R01-DK-50277 and Diabetes Research and
Training Center Grant P60-DK-20593.
Current address for Dr. Yoshiharu Koyama: Department of Critical Care
Medicine, Tokyo Medical and Dental University, 5-45, Yushima
1-chome, Bunkyo-ku, Tokyo 113-8519, Japan.
Address for reprint requests and other correspondence: D. Wasserman, Dept. of Molecular Physiology and Biophysics, Vanderbilt Univ. School of Medicine, Light Hall Rm. 702, Nashville, TN 37232-0615 (E-mail: david.wasserman{at}mcmail.vanderbilt.edu).
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.
10.1152/ajpendo.00370.2001
Received 16 August 2001; accepted in final form 12 January 2002.
 |
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