Exercise-Stimulated Glucose Turnover in the Rat Is Impaired by Glucosamine Infusion
Philip D.G. Miles,
Katsuya Higo, and
Jerrold M. Olefsky
From the Departments of Surgery (P.D.G.M.) and Medicine (K.H., J.M.O.),
University of California-San Diego; the Division of Endocrinology and
Metabolism (J.M.O.), San Diego VA Medical Center, San Diego; and the Whittier
Diabetes Institute (J.M.O.), La Jolla, California.
Address correspondence and reprint requests to Dr. Philip D.G. Miles,
Department of Surgery, UCSD Medical Center, 200 West Arbor Dr., San Diego, CA
92103-8400. E-mail:
pmiles{at}ucsd.edu
.
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ABSTRACT
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The infusion of glucosamine causes insulin resistance, presumably by
entering the hexosamine biosynthetic pathway; it has been proposed that this
pathway plays a role in hyperglycemia-induced insulin resistance. This study
was undertaken to determine if glucosamine infusion could influence
exercise-stimulated glucose uptake. Male SD rats were infused with glucosamine
at 0.1 mg · kg-1 · min-1 (low-GlcN group),
6.5 mg · kg-1 · min-1 (high-GlcN group),
or saline (control group) for 6.5 h and exercised on a treadmill for 30 min
(17 m/min) at the end of the infusion period. Glucosamine infusion caused a
modest increase in basal glycemia in both experimental groups, with no change
in tracer-determined basal glucose turnover. During exercise, glucose turnover
increased
2.2-fold from 46 ± 2 to 101 ± 5 µmol ·
kg-1 · min-1 in the control group. Glucose
turnover increased to a lesser extent in the glucosamine groups and was
limited to 88% of control in the low-GlcN group (47 ± 2 to 90 ±
3 µmol · kg-1 · min-1; P <
0.01) and 72% of control in the high-GlcN group (43 ± 1 to 73 ±
3 µmol · kg-1 · min-1; P <
0.01). Similarly, the metabolic clearance rate (MCR) in the control group
increased 72% from 6.1 ± 0.2 to 10.5 ± 0.7 ml ·
kg-1 · min-1 in response to exercise. However,
the increase in MCR was only 83% of control in the low-GlcN group (5.2
± 0.5 to 8.7 ± 0.5 ml · kg-1 ·
min-1; P < 0.01) and 59% of control in the high-GlcN
group (4.5 ± 0.2 to 6.2 ± 0.3 ml · kg-1
· min-1; P < 0.01). Neither glucosamine infusion
nor exercise significantly affected plasma insulin or free fatty acid (FFA)
concentrations. In conclusion, the infusion of glucosamine, which is known to
cause insulin resistance, also impaired exercise-induced glucose uptake. This
inhibition was independent of hyperglycemia and FFA levels.
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INTRODUCTION
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Chronic hyperglycemia is the cardinal metabolic feature of diabetes and can
adversely affect glucose homeostasis by interfering with insulin action and/or
ß-cell function (1).
Studies in humans have shown that experimental hyperglycemia or
diabetes-associated hyperglycemia can cause insulin resistance or exacerbate
an underlying insulin-resistant state
(2). This finding is supported
by animal and in vitro studies that directly show that hyperglycemia can cause
impaired insulin action in vitro and insulin resistance in vivo
(1,3,4).
The mechanism by which hyperglycemia causes insulin resistance remains
incompletely understood, but several studies have implicated the hexosamine
biosynthesis pathway
(5,6,7,8).
This hypothesis holds that hyperglycemia, by way of mass action, leads to
increased flux of glucose carbons through the hexosamine pathway
(9). This occurs by
glutamine:fructose-6-phosphate amidotransferase (GFAT)-mediated conversion of
fructose-6-phosphate (F6P) to glucosamine-6-phosphate (G6P), with subsequent
metabolism of G6P through the hexosamine pathway. Distal metabolic products of
this pathway in some way may lead to decreased insulin-stimulated GLUT4
translocation and subsequent reduction in glucose metabolism
(10). Glucosamine also
increases flux through the hexosamine pathway; however, it bypasses GFAT and
enters the pathway as G6P. Indeed, the infusion of glucosamine has been shown
to produce insulin resistance in rats
(6,11,
which is not additive to the effect of hyperglycemia
(6). Furthermore, mice who
overexpress GFAT are insulin resistant
(12).
In addition to insulin, exercise increases the uptake of glucose into
contracting muscle by stimulating the movement of GLUT4 transporter-containing
vesicles to the plasma membrane and transverse tubules
(13). Despite being
insulin-resistant, obese rats have normal increases in peripheral glucose
uptake in response to moderate exercise
(14,15),
and similar findings have been found in obese type 2 diabetic subjects
(16). These findings are not
unexpected, considering it is generally accepted that exercise and insulin use
different intracellular signaling pathways and recruit distinct GLUT4
transporters from different intracellular vesicular pools
(13).
Because exercise and insulin use distinct pathways to augment glucose
transport, it is not known whether stimulation of the hexosamine pathway can
influence exercise-stimulated glucose uptake. To examine this question, we
studied the effects of glucosamine infusion on exercise-stimulated glucose
disposal in male SD rats.
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RESEARCH DESIGN AND METHODS
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Animals and training. Male SD rats (Harlan, Indianapolis, IN)
weighing 200-224 g were housed individually under controlled light/dark (12/12
h) and temperature conditions and had free access to food and water. The
animals were trained daily for 4 days to run on a treadmill (in-house design
and construction) for 30 min at 17 m/min and 0% slope. The animals then
underwent surgery during which catheters were implanted. Experiments were
performed on the animals 4-5 days after surgery with a single training bout in
between. All procedures were in accordance with the Guide for the Care and
Use of Laboratory Animals of the National Institutes of Health and
approved by the Animal Subjects Committee of the University of California, San
Diego.
Surgery. One catheter was placed in the left jugular vein
(Micro-Renathane MRE-033,0.033 in OD and 0.014 in ID; Braintree Scientific,
Braintree, MA) and another was placed in the left carotid artery under general
anesthesia. The anesthetic cocktail consisted of ketamine HCl (50 mg/kg)
(Aveco, Fort Dodge, IA), acepromazine maleate (1 mg/kg) (Aveco), and xylazine
(4.8 mg/kg) (Lloyd, Shenandoah, IA) given intramuscularly. Catheters were
tunneled subcutaneously, exteriorized at the back of the neck, filled with
heparinized saline, and flushed every other day to maintain patency.
Ampicillin (1 mg/kg) (Aveco) was given prophylactically at the time of
surgery. The jugular and carotid catheters were used for infusion and blood
sampling, respectively, during the subsequent experiment.
Exercise experiment. The jugular line was connected to an infusion
pump via swivel 4-5 days after catheter placement, allowing the animals to
move freely in a circular container. The animals were infused (0.5 ml/h) for
6.5 h with saline or glucosamine (0.1 or 6.5 mg · kg-1
· min-1). Glucose turnover during exercise was assessed with
the infusion of tritiated glucose. A priming injection (2.5 µCi/0.5 ml) and
constant infusion (0.04 µCi/min) of D-[3-3-H]glucose
(DuPont-NEN, Boston, MA) was started 5 h into the saline or glucosamine
infusion. Tracer glucose was diluted to 5 µCi/ml in saline containing 100
mg/dl unlabeled D-glucose (Mallinckrodt, Paris, KY) as carrier and 200 mg/dl
sodium benzoate (Mallinckrodt) as preservative. The animals were then
transferred onto the treadmill. After a 1-h tracer equilibration period at
hour 6, the animals were run for 30 min at 17 m/min and 0% slope. Blood
samples (0.5 ml) were collected in heparinized microtubes immediately before
exercise and at the end of exercise for determination of plasma glucose,
glucose-specific activity, insulin, and free fatty acid (FFA).
All blood samples were immediately stored at 4°C. Blood was centrifuged
within 20 min of collection and the resultant plasma was stored at
-20°C.
Assays. Plasma glucose concentration was measured with a YSI 23A
glucose analyzer (Yellow Springs Instruments, Yellow Springs, OH). Plasma for
determination of [3-3H]glucose was deproteinized with perchloric
acid and assayed as previously described
(2). Insulin was measured by
radioimmunoassay with a double-antibody immunoprecipitation technique as
previously described (17). FFA
was assayed according to standardized technique
(18).
Calculations. Pre-exercise and exercise glucose turnover was
calculated using the Steele equation for steady-state conditions
(19). Specifically, hepatic
glucose production (HGP) and glucose disposal rate (GDR) were assumed to be
equal by 30 min of exercise (see APPENDIX for the validation study). The
metabolic clearance rate (MCR) was calculated by normalizing the glucose
turnover with the glucose concentration
(20). Data are means ±
SE. Statistical analysis was performed using a two-way analysis of variance
for unbalanced data and significance was assumed at P < 0.05.
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RESULTS
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We studied three groups of exercised animals: 1) saline infused
rats (control group; n = 9), 2) rats infused with 0.1 mg
· kg-1 · min-1 glucosamine (low-GlcN
group; n = 6), and 3) rats infused with 6.5 mg ·
kg-1 · min-1 glucosamine (high-GlcN group;
n = 11). The weights of the control (237 ± 9 g), low-GlcN (239
± 5 g), and high-GlcN (245 ± 6 g) groups were all similar
(NS).
Pre-exercise period. As seen in
Fig. 1A, the infusion
of glucosamine for 6 h caused a modest increase in plasma glucose
concentration in the low-GlcN (9.0 ± 0.3 mmol/l; P < 0.01)
and high-GlcN (9.6 ± 0.3 mmol/l; P < 0.01) groups compared
with the saline-infused control group (7.5 ± 0.1 mmol/l). Glucosamine
administration did not significantly alter tracer-determined glucose turnover
compared with the control group value of 46 ± 2 µmol ·
kg-1 · min-1
(Fig. 1B). However,
the higher plasma glucose level can increase glucose disposal by mass action.
Therefore, when considering the prevailing glucose concentration, MCR
(Fig. 1C) decreased in
both the low-GlcN (5.2 ± 0.5 ml · kg-1 ·
min-1; P < 0.05) and high-GlcN (4.5 ± 0.2 ml
· kg-1 · min-1; P < 0.01)
groups in comparison with the control group (6.1 ± 0.2 ml ·
kg-1 · min-1). The glucosamine infusions had no
significant effects on plasma insulin or FFA concentrations
(Fig. 2).

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FIG. 1. The effect of glucosamine infusion (low-GlcN group, 0.1 mg ·
kg-1 · min-1; high-GlcN group, 6.5 mg ·
kg-1 · min-1) and saline infusion (control group)
on plasma glucose (A), glucose turnover (B), and MCR
(C) before and during exercise. Pre-exercise values were measured
after 6 h of glucosamine infusion, and exercise values were measured 30 min
into exercise and after 6.5 h of glucosamine infusion. Data are means ±
SE. *Significantly different from control group (P <
0.05); #significantly different from the corresponding pre-exercise group
(P < 0.05).
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FIG. 2. The effect of glucosamine infusion (low-GlcN group, 0.1 mg ·
kg-1 · min-1; high-GlcN group, 6.5 mg ·
kg-1 · min-1) and saline infusion (control group)
on plasma insulin (A) and FFA (B) before and during
exercise. Pre-exercise values were measured after 6 h of glucosamine infusion,
and exercise values were measured 30 min into exercise and after 6.5 h of
glucosamine infusion. Data are means ± SE. *Significantly
different from control group (P < 0.05); #significantly different
from corresponding pre-exercise group (P < 0.05).
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End of exercise period. The 30 min of exercise led to a marked
increase in plasma glucose in the control group (7.5 ± 0.1 to 9.6
± 0.4 mmol/l; P < 0.001), and further exacerbated the
hyperglycemia in the low-GlcN group (9.0 ± 0.3 to 10.2 ± 0.3
mmol/l; P < 0.05) and high-GlcN group (9.6 ± 0.3 to 11.6
± 0.4 mmol/l; P < 0.01) groups
(Fig. 1A). As
expected, glucose turnover (Fig.
1B) increased 2.2-fold in response to exercise, from 46
± 2 to 101 ± 5 µmol · kg-1 ·
min-1 in the control group. On the other hand, the effect of
exercise in stimulating GDR was blunted in the glucosamine-infused animals.
Thus, in the low-GlcN group, GDR was only 90 ± 3 µmol ·
kg-1 · min-1, which was 12% less (P <
0.05) than in the control group, and was only 73 ± 3 µmol ·
kg-1 · min-1 in the high-GlcN group, which was
28% less (P < 0.01) than in the control group. Similarly, as seen
in Fig. 1C, the MCR in
the control group increased markedly from 6.1 ± 0.2 to 10.5 ±
0.7 ml · kg-1 · min-1 in response to
exercise, whereas this effect was blunted in the glucosamine groups. MCR
values were 8.7 ± 0.5 (P < 0.01) and 6.2 ± 0.3 ml
· kg-1 · min-1 (P < 0.01) in
the low- and high-GlcN groups, respectively. The glucosamine-induced
inhibition of GDR was even more pronounced if one examines only the
exercise-induced increment in GDR. With this analysis, the exercise-induced
increments in MCR were decreased by 21 and 61% in the low- and high-GlcN
groups, respectively. Exercise and the combination of exercise and glucosamine
infusion had no effect on plasma insulin or FFA concentrations
(Fig. 2).
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DISCUSSION
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Hyperglycemia can cause a secondary state of insulin resistance in vitro
(1), in animals
(3,4),
and in humans (2); it has been
proposed that one mechanism underlying this phenomenon involves the hexosamine
pathway
(5,6,7,8).
According to this hypothesis, hyperglycemia prevents increased glucose uptake
by increasing flux through the hexosamine pathway with the production of
distal hexosaminoglycan products, which impair insulin signaling. Glucosamine
enters this pathway distal to the GFAT enzymatic step, and administration of
glucosamine to animals causes in vivo insulin resistance, reportedly by
inhibiting early steps in insulin-stimulated glucose transport
(10). Acute exercise
stimulates GLUT4 translocation and glucose transport by mechanisms that are
independent of the insulin-signaling pathway
(13). In previous studies, we
found that glucosamine infusion markedly inhibited insulin-stimulated GDR in
rats (11), and in the current
study we found that glucosamine administration has a similar effect on
impairing the effects of acute exercise to enhance GDR.
From these results, several conclusions and speculations are possible.
First, if glucosamine causes insulin resistance by interfering with the
initial steps of insulin action, then, because exercise stimulates GDR by a
separate pathway, one would not expect glucosamine infusions to inhibit the
effect of exercise on GDR. Therefore, glucosamine must either have two
separate and independent effects, one in the insulin and the other in the
exercise pathway, or work at a distal step in glucose transport stimulation
common to both. In this latter regard, it has been shown that glucosamine can
cause cellular ATP depletion, which inhibits distal steps of GLUT4 trafficking
(21). Such an effect of
glucosamine is unlikely to be representative of the mechanisms by which
hyperglycemia causes insulin resistance.
It is well known that obese type 2 diabetic patients are insulin resistant
(22,23),
and it has also been shown that the acute effects of exercise to augment GDR
are normal in these patients
(16). In other words, they are
insulin resistant, but not exercise resistant, with respect to stimulation of
GDR. Because glucosamine inhibits the effects of insulin and exercise on GDR,
insofar as glucosamine mimics activation of the hexosamine pathway, our
results suggest that increased flux through the hexosamine pathway is probably
not a major mechanism of in vivo hyperglycemia-induced insulin resistance.
Circulating FFAs were not a factor in the impairment of exercise-stimulated
glucose turnover. FFAs can inhibit glucose metabolism via the glucose/fatty
acid cycle (24), but, as seen
in Fig. 2, they did not change
with glucosamine infusion or exercise in the current study.
Our results compare well with those of previous studies in which the
influence of glucosamine on glucose metabolism was examined. In this study,
glucosamine was infused at 0.1 and 6.5 mg · kg-1 ·
min-1, and saline infusion served as the control. Rossetti et al.
(6) previously demonstrated
that these infusion rates represent the minimal and maximal effective dosages
that induce insulin resistance, respectively, and thus they were chosen in
this study. Although we did not directly measure insulin sensitivity, the
significant decline seen in pre-exercise MCR, for which MCR is a measure of
glucose turnover normalized for plasma glucose, was evidence of insulin
resistance at both dosages. In our hands, the infusion of glucosamine at 0.1
and 6.5 mg · kg-1 · min-1 resulted in a 17
and 41% reduction in exercise-stimulated MCR, respectively, compared with the
rates in saline-infused animals. These impairments of MCR are comparable with
those previously observed with insulin-stimulated glucose uptake, when glucose
uptake was reduced by 18% in animals infused with 0.1 mg ·
kg-1 · min-1 glucosamine and 30% in those infused
with 6.5 mg · kg-1 · min-1 glucosamine
(6).
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APPENDIX
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This study was undertaken to determine when glucose turnover reaches steady
state during exercise and whether a single blood sample taken during this
period can be used to measure it. Animals were handled as described above with
the following differences. The animals were fasted for 6 h before exercise
rather than being infused with saline or glucosamine. Blood samples were
collected at 20, 25, 30, and 35 min during exercise for determination of
plasma glucose and glucose-specific activity. Glucose concentration (G) and
specific activity (SA) were smoothed using the optimal segments program
(25) for the purpose of
estimating dSA/dt and dG/dt. HGP and GDR were calculated using the Steele
Equation for non-steady-state conditions
(19). Glucose turnover was
calculated using the Steele equation for steady-state conditions
(19), which assumes HGP and
GDR are equal.
A total of six animals completed the study. As seen in
Fig. 3, HGP and GDR were equal
and remained constant toward the end of the exercise period; that is, glucose
turnover was in steady state by the end of exercise. Not surprisingly, the
calculation of glucose turnover for non-steady-state conditions and assuming
steady-state conditions (dSA/dt and dG/dt = 0) produced similar values.
Consequently, the estimation of glucose turnover using a single blood sample
taken 25-35 min into moderate exercise (17 m/s, 0% slope) is valid.

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FIG. 3. A comparison of glucose turnover in response to moderate exercise
estimated using Steele's equation for non-steady-state conditions (HGP, GDR)
and steady state (SS) conditions, which assumes HGP and GDR are equal. A total
of six animals were tested.
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It should be noted, however, that these validation experiments were
successfully completed in only 6 of 17 animals tested. In 66% of the animals,
multiple blood sampling during exercise was not possible, or the action of
drawing blood caused the animal to repeatedly stop running. Therefore, we
settled on a single blood sample collected at 30 min of exercise to estimate
glucose turnover.
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ACKNOWLEDGMENTS
|
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This study was supported in part by a grant from the National Institutes of
Health (DK-33651), the Veterans Administration Research Service, the
Department of Veterans Affairs, and the Whittier Diabetes Institute.
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FOOTNOTES
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F6P, fructose-6-phosphate; FFA, free fatty acid; G6P,
glucosamine-6-phosphate; GDR, glucose disposal rate; GFAT,
glutamine:fructose-6-phosphate amidotransferase; HGP, hepatic glucose
production; MCR, metabolic clearance rate.
Received for publication April 11, 2000
and accepted in revised form September 14, 2000
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