Endocrinology Vol. 140, No. 3 1151-1157
Copyright © 1999 by The Endocrine Society
Mechanism of Hexosamine-Induced Insulin Resistance in Transgenic Mice Overexpressing Glutamine:Fructose-6-Phosphate Amidotransferase: Decreased Glucose Transporter GLUT4 Translocation and Reversal by Treatment with Thiazolidinedione1
Robert C. Cooksey2,
Leon F. Hebert, Jr.2,
Ju-Hong Zhu,
Perisco Wofford,
W. Timothy Garvey and
Donald A. McClain
Departments of Medicine of the University of Mississippi
Medical Center (D.A.M., R.C.C., L.F.H., P.W.), Jackson, Mississippi
39216 and the Medical University of South Carolina (J.-H.Z., W.T.G.)
Charleston, South Carolina 29425; and the Veterans Affairs
Medical Centers at Jackson, Mississippi 39216 (D.A.M., R.C.C.) and
Charleston, South Carolina 29425 (W.T.G.)
Address all correspondence and requests for reprints to: Donald A. McClain, Division of Endocrinology, University of Mississippi Medical Center, 2500 North State Street, Jackson, Mississippi 39216-4505. E-mail: dam{at}fiona.umsmed.edu
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Abstract
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Hexosamines have been hypothesized to mediate aspects of glucose
sensing and toxic effects of hyperglycemia. For example, insulin
resistance results when the rate-limiting enzyme for hexosamine
synthesis, glutamine:fructose-6-phosphate amidotransferase (GFA), is
overexpressed in muscle and adipose tissue of transgenic mice. The
glucose infusion rates required to maintain euglycemia at insulin
infusion rates of 0.5, 2, 15, and 20 mU/kg·min were 39–90% lower in
such transgenic mice, compared with their control littermates
(P 
0.01). No differences were observed in
hepatic glucose output, serum insulin levels, or muscle ATP levels.
Uptake of 2-deoxyglucose, measured under conditions of
hyperinsulinemia, was significantly lower in transgenic hindlimb
muscle, compared with controls (85.9 ± 17.8 vs.
166.8 ± 15.1 pmol deoxyglucose/g·min). The decrease in glucose
uptake by transgenic muscle was associated with a disruption in the
translocation of the insulin-stimulated glucose transporter GLUT4.
Fractionation of muscle membranes on a discontinuous sucrose gradient
revealed that insulin stimulation of control muscle led to a 28.8%
increase in GLUT4 content in the 25% fraction and a 61.2% decrease in
the 35% fraction. In transgenic muscle, the insulin-stimulated shifts
in GLUT4 distribution were inhibited by over 70%. Treatment of the
transgenic animals with the thiazolidinedione troglitazone
completely reversed the defect in glucose disposal without changing GFA
activity or the levels of uridine
5'-diphosphate-N-acetylglucosamine. Overexpression of
GFA in skeletal muscle thus leads to defects in glucose transport
similar to those seen in type 2 diabetes. These data support the
hypothesis that excess glucose metabolism through the hexosamine
pathway may be responsible for the diminished insulin sensitivity and
defective glucose uptake that are seen with hyperglycemia.
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Introduction
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SKELETAL MUSCLE is responsible for the
majority of insulin-stimulated glucose uptake (1). Insulin resistance
and decreased insulin-stimulated glucose use are hallmarks of type 2
diabetes mellitus and can be seen as a result of hyperglycemia in type
1 diabetes, as well (2, 3, 4). The mechanism of these effects is unknown.
The hexosamine biosynthetic pathway has been shown to play a role in
some of the adverse regulatory effects of hyperglycemia (5). For
example glucosamine infusion in rats causes insulin resistance in
skeletal muscle by inhibiting insulin’s ability to stimulate the
translocation of the insulin-stimulated glucose transporter (GLUT4)
from intracellular pools to the plasma membrane (6, 7). Glucosamine
also causes insulin resistance for glycogen synthesis (8).
We have previously demonstrated that overexpression of
glutamine:fructose-6-phosphate amidotransferase (GFA), the first and
rate-limiting enzyme of the hexosamine biosynthetic pathway, in muscle
and adipose tissue of transgenic mice leads to increased hexosamine
levels and causes insulin resistance in vivo (9). The
current study was undertaken to explore further the relationship of the
defects in glucose uptake seen in this transgenic model to those of
type 2 diabetes, in terms of the mechanism for the decreased glucose
uptake and its reversal by the peroxisome proliferator-activated
receptor (PPAR)
agonist troglitazone. The results are
consistent with the hypothesis that excess glucose metabolism through
the hexosamine pathway may be responsible for the diminished insulin
sensitivity and defective glucose uptake that are seen in
hyperglycemia. Furthermore, the defects in glucose transport seen in
the transgenic mice are very similar to those seen in human type 2
diabetes.
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Materials and Methods
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Animals
The creation of transgenic mice overexpressing human GFA in
skeletal muscle through the use of the GLUT4 promoter/enhancer has been
previously described (9). All animals used for experiments were 3- to
6-month-old littermates resulting from the breeding of a single line of
GFA overexpressing mice. Heterozygote transgenic animals, bred on a
C57Bl6 background, were crossed to nontransgenic C57Bl6 mice, resulting
in litters consisting of approximately 50% heterozygote transgenic
animals and an equal number of nontransgenic littermates that were used
as controls. Animals were housed in a room maintained at 23 C with a
fixed 12-h light, 12-h dark cycle and given free access to rat chow and
water. Experimental protocols were approved by the Animal Use Committee
of the Veterans Affairs Medical Center.
To examine the effects of troglitazone, age- and
weight-matched heterozygote transgenic and control mice were treated
daily for 30 days with either 10 mg troglitazone mixed in
100 mg of peanut butter or with 100 mg of peanut butter alone. Five
mice in each group were then studied by the hyperinsulinemic euglycemic
clamp technique, after which the animals were killed and their hindlimb
muscles harvested for analysis of GFA activity and uridine
5'-diphosphate (UDP)-N-acetylglucosamine levels.
Determination of glucose infusion rate and hepatic glucose output
(HGO)
All experiments were performed in awake, weight-matched,
nonsedated transgenic and littermate control mice using a euglycemic
clamp technique previously described (9). Catheters were implanted into
the right internal jugular vein. The animals were allowed to recover
from surgery for 1 day and were then fasted 18 h before the
experiment. [3-3H]glucose (New England Nuclear, Boston,
MA) was infused throughout the clamp experiment to determine the
glucose turnover rate. A priming dose of 0.33 µCi (19 Ci/mmol) in
11.2 µl saline (0.9%) was infused, followed by continuous infusion
at a rate of 0.08 µCi/min (2.5 µl/min) for the duration of the
experiment. Animals were infused with recombinant human insulin
(HumulinR, Eli Lilly & Co., Indianapolis, IN) at a rate of
2, 15, or 20 mU/kg·min, while 50% dextrose was infused by a variable
infusion pump (Harvard Apparatus Inc., South Natick, MA). Whole-blood
samples (3 µl) were collected every 5–10 min from tail bleeds and
measured by the glucose oxidase method (Glucometer Elite, Bayer Corp., Tarrytown, NY). Blood glucose concentrations and glucose
infusion rates were clamped at steady state for a minimum of 20 min.
Serum samples were then taken for calculating insulin levels and HGO.
Insulin concentrations were measured by RIA using porcine standards
(Binax, Portland, ME); this assay detects both human and rodent
insulin. The rate of glucose appearance (or glucose turnover rate) was
calculated by dividing the [3-3H]glucose infusion rate
(dpm/kg·min) by the mean serum glucose specific activity (dpm/mg
glucose). The rate of HGO was calculated by subtracting the glucose
infusion rate from the glucose turnover rate. Isotopically determined
glucose disposal rates (GDRs) were calculated by subtracting the HGO
from the glucose infusion rate.
Determination of skeletal muscle glucose uptake, GFA activity,
UDP-N-acetylglucosamine levels, and ATP levels
Mice were catheterized, allowed to recover from surgery the
following day, and fasted 18 h before the experiment. With no
priming dose or continuous infusion of 3H-glucose, the
animals were infused with recombinant human insulin at a rate of 20
mU/kg·min, while 50% dextrose was infused by a variable infusion
pump. After the animals’ serum glucose levels were stabilized (minimum
of 10 min), a bolus injection of
2-deoxy-D-[1-3H]glucose
([3H]2-DOG) and [U-14C]sucrose (200 pmol of
each, 11 Ci/mmol and 667 mCi/mmol, respectively; Amersham, Arlington
Heights, IL) was administered. The clamp was continued for an
additional 10 min, after which time the animal was killed, blood was
collected, and the triceps surae group from each hind limb was
extracted, weighed, and frozen in liquid nitrogen. The triceps surae
group was selected because its muscle fiber composition is
representative of total hindlimb fiber composition (10). Muscle samples
were processed as described by Brozinick et al. (11). Muscle
samples were homogenized in 2 ml of 10% trichloroacetic acid (TCA) at
4 C. Muscle homogenates and serum samples (20 µl serum with 40 µl
10% TCA) were centrifuged in a microcentrifuge at 14,000 x
g for 10 min. Radioactivity in duplicate samples of the
muscle and serum supernatants (300 µl of muscle supernatant and 15
µl of the serum supernatant) was measured by scintillation counting.
The accumulation of intracellular [3H]2-DOG was
calculated by subtracting the amount in the extracellular space from
the total in the muscle samples, using the facts that glucose and
sucrose will both diffuse in the extracellular space but sucrose
will not be transported into cells. Thus, intracellular glucose =
total muscle glucose - extracellular glucose, where extracellular
glucose = (serum glucose) · (muscle sucrose) · (serum
sucrose)-1.
GFA activity and UDP-N-acetylglucosamine levels were assayed
as described (9). ATP measurements were performed on triceps surae
group muscle that had been homogenized and sonicated in 12% TCA, using
a kit from Sigma Chemical Co. (St. Louis, MO), based on
the principle that ATP levels can be determined by measuring the change
in absorbance at 340 nm that takes place when NADH is oxidized to
NAD.
Membrane subfractionation of skeletal muscle
Hindlimb muscle from transgenic and control mice that had been
either fasted (basal) or subjected to a hyperinsulinemic-euglycemic
clamp (insulin-stimulated) was rapidly isolated, frozen in liquid
nitrogen, and stored at -80 C. The muscle tissue was placed in Tris
buffer (20 mM Tris-base, 0.25 M sucrose, 0.2
mM EDTA, 40 mM phenylmethylsulfonylfluoride, 5
µg/ml leupeptin, 5 µg/ml aprotinin, and 1 µg/ml pepstatin), pH
7.4, at 4 C. Subfractionation of muscle membranes was as described by
Baron and colleagues (7), whose procedure was modified from that of
Klip and colleagues (12, 13). The muscle was homogenized with a
Polytron tissue disrupter (Brinkmann Instruments, Inc.,
Westbury, NY) using five 5-sec bursts at a setting of 5 and then with
10 up-and-down strokes of a motor-driven Teflon pestle in a glass
homogenization tube (Thomas Scientific, Philadelphia, PA). The
homogenate was centrifuged at 1,000 x g for 10 min,
and the supernatant was saved. The resulting pellet was resuspended in
the buffer and rehomogenized with the glass homogenization tube and
Teflon pestle (as described above), and the supernatant was combined
with the first supernatant and centrifuged at 9,000 x
g for 10 min. The resulting supernatant was then centrifuged
at 190,000 x g for 60 min; the resuspended pellet
constituted the total postnuclear membrane fraction. These membranes
were then applied to a discontinuous sucrose gradient containing 25%,
30%, and 35% sucrose (wt/vol) solutions and was centrifuged at
40,000 x g for 16 h in a swinging-bucket rotor.
Membranes were collected atop each of the sucrose gradients,
resuspended in Tris buffer, pelleted by centrifugation at 190,000
x g for 60 min, and resuspended in Tris buffer. All
suspensions of membrane subfractions were kept at -80 C for subsequent
immunoblot analyzes. To monitor membrane subfractionation, we measured
5'nucleotidase activity using the method of Avruch and Wallach (14),
oubain-inhibitable Na+/K+ ATPase activity, as
described previously (15), and immunoreactive phospholemman (16, 17) as
markers for sarcolemma in total membranes and membrane
subfractions.
Immunoblot analysis
Thirty micrograms of membrane protein were solubilized in
Laemmli sample buffer (18), electrophoresed by SDS-PAGE on 1.5-mm slab
gels, and transferred to nitrocellulose filters (19). Immunological
detection of GLUT4 was accomplished as previously described (20).
Nitrocellulose filters were incubated with affinity-purified rabbit
antiserum (1:1000 dilution) specific for the COOH-terminal segment (12
amino acids) of rat GLUT4 (21) (East Acres Biologicals,
Southbridge, MA), followed by 125I-protein A. Detection of
phospholemman was accomplished by incubating filters with
affinity-purified rabbit antiserum raised against canine phospholemman
(16, 17) (a gift of Dr. Larry Jones, Indiana University), followed by
125I-protein A. Quantitation of GLUT4 levels was
accomplished by excising the GLUT4 band and measuring radioactive
counts. Phospholemman was measured with scanning densitometry (Bio-Rad,
Richmond, CA) of autoradiographs. For measurement of both GLUT4 and
phospholemman, background measurements were subtracted from signal, to
obtain relative levels of the immunoreactive protein; and measurements
were consistently in the range where the relationship between
increasing amounts of membrane protein and signal was linear.
Statistical analysis
All values are presented as mean ± SEM.
Student’s t test was performed for comparisons of means
between two groups.
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Results
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Glucose infusion rates and HGO during the euglycemic clamp
Nonsedated weight-matched transgenic and control littermates
(22.5 ± 1.1 and 22.2 ± 1.6 µg, respectively) were clamped
at a blood glucose of 8.3 ± 0.2 mM while being
infused with insulin at a constant rate of 0.5, 2, 15, or 20
mU/kg·min. At the end of the clamp, the glucose infusion rate was
calculated, and blood was collected to calculate HGO and determine
insulin levels. HGO was not determined for mice clamped at the insulin
infusion rate of 0.5 mU/kg·min, because of experimental limitations.
Calculation of the glucose infusion rates shows that transgenic mice
required a lower level of infused glucose to maintain similar blood
glucose levels, compared with their control littermates (Fig. 1A
). The transgenic mice required a 90,
45, 39, and 40% reduction of the glucose infusion rate to maintain
euglycemia during the clamp at insulin infusion rates of 0.5, 2, 15,
and 20 mU/kg·min, respectively. HGO did not differ between transgenic
and control mice under basal conditions and during the euglycemic clamp
with insulin infusion rates of 2, 15, and 20 mU/kg·min (Fig. 1B).
Serum insulin levels also did not differ between transgenic and control
mice during the euglycemic clamp (Fig. 1C).
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Figure 1. Euglycemic clamp of transgenic and control mice.
A, Glucose infusion rates, to maintain a constant blood glucose value
of 8.3 ± 0.2 mM during the clamp; B, HGO during the
clamp; C, serum insulin levels during the clamp. Shown is mean ±
SEM; n = 3/group; *, P < 0.01.
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Decreased glucose uptake by skeletal muscle during the
hyperinsulinemic-euglycemic clamp
Weight-matched transgenic and control mice (23.9 ± 2.1 and
25.4 ± 2.4 µg, respectively) were clamped at a blood glucose of
7.9 ± 0.7 mM during constant insulin infusion of 20
mU/kg·min, without the priming dose or continual infusion of
[3-3H]glucose. Ten minutes after equilibration of glucose
levels, [3H]2-DOG and [14C-U]sucrose were
administered for 10 min. After a further 10 min of the clamp procedure,
GDRs were calculated, and serum and hindlimb triceps surae muscle were
collected to measure [3H]2-DOG uptake. Glucose uptake was
determined by correcting total muscle [3H]2-DOG for
interstitial accumulation, assessed by measuring
[14C-U]sucrose accumulation. Fig. 2A shows the GDRs for the transgenic and
control mice during the clamp studies. Transgenic mice had a GDR that
was 35% lower than that of the control mice. Fig. 2B shows that
[3H]2-DOG uptake by skeletal muscle was decreased by 49%
in transgenic mice, compared with control littermates.
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Figure 2. Insulin-stimulated uptake of
[3H]2-DOG by skeletal muscle. A, GDRs during a
hyperinsulinemic euglycemic clamp (20 mU/kg·min); B, uptake of
[3H]2-DOG by the triceps surae muscle group of mice
during a hyperinsulinemic euglycemic clamp (20 mU/kg·min). Shown is
mean ± SEM; n = 4/group; *,
P < 0.05.
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ATP levels in muscle were also assayed after the hyperinsulinemic clamp
studies. There was no significant difference in the ATP level in
transgenic muscle (0.70 ± 0.18 µmol/g muscle), compared with
control muscle (0.68 ± 0.13 µmol/g muscle, n = 6 per
group).
In vitro studies of skeletal muscle GLUT4 distribution
Hindlimb skeletal muscle from weight-matched, fasted transgenic
and control mice (26.4 ± 0.8 and 25.2 ± 1.8 µg,
respectively) was harvested either in the basal state or after a clamp
at 9.3 ± 0.3 mM glucose with an insulin dose of 20
mU/kg·min. The muscle was subjected to a membrane subfractionation
protocol employing centrifugation on discontinuous sucrose density
gradients. GLUT4 levels in the various subcellular fractions were
quantitated by PAGE of membrane proteins, followed by immunoblot
analysis. The results of these analyzes are shown in Table 1. Total GLUT4 content did not differ
between transgenic and control muscle. GLUT4 distribution also did not
differ in the basal state between transgenic and control muscle, but
differences did emerge with insulin stimulation. After insulin
stimulation of control muscle, GLUT4 distribution increased in the 25%
fraction by 28.8 ± 1.9% (P < 0.05) and
decreased in the 35% fraction by 61.2 ± 1.1% (P
< 0.001). Insulin led to smaller and statistically insignificant
changes in GLUT4 distribution in transgenic muscle, increasing in the
25% fraction by 8.5 ± 5.0% and decreasing in the 35% fraction
by 7.5 ± 5.3%. The differences in insulin-induced GLUT4
redistribution between the transgenic and control muscles were
significant in the 25% fractions (P < 0.05) and in
the 35% fractions (P < 0.001). No differences,
comparing either transgenic to control or insulin-stimulated to basal,
were noted in the GLUT4 content in the 30% fraction. Two sarcolemmal
markers, 5'-nucleotidase activity and immunoreactive phospholemman,
were measured and found to be greatest in the 25% fraction (data not
shown). These data are consistent with previous studies and have been
interpreted to reflect insulin-mediated translocation of GLUT4 from
intracellular membranes in the 35% sucrose fraction to sarcolemma
retrieved in the 25% fraction (7, 12, 13).
Reversal of insulin resistance for glucose disposal by
troglitazone
Nonsedated weight-matched transgenic and control mice (25.6
± 1.8 and 24.0 ±1.5 g, respectively) were treated with
troglitazone (10 mg/day for 30 days) or vehicle. The mice
were then subjected to a hyperinsulinemic euglycemic clamp with insulin
(20 mU/kg·min) and 50% glucose infusion. As shown in Fig. 3, the decrease in glucose disposal in
the transgenic animals was completely reversed by
troglitazone treatment (P < 0.001, n
= 5/group). The drug was without a significant effect in the control
animals.
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Figure 3. GDRs in control and transgenic mice treated with
troglitazone. Animals were age- and weight-matched (90
days; 24.1 ± 1.5, 25.6 ±1.1 µg, respectively). Shown is
mean ± SEM; n = 5/group; *,
P < 0.001
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To determine whether the effect of troglitazone was distal
to the generation of hexosamines, the levels of GFA and
UDP-N-acetylglucosamine were next measured in the muscle of
the control and drug-treated animals (Fig. 4). As had been previously demonstrated,
GFA activity was higher in the transgenic animals, compared with
controls (P < 0.05 for the nondrug-treated animals,
P < 0.03 for the drug-treated animals). However,
troglitazone had no effect on GFA activity, either in
control or transgenic animals. Similarly, the principal end product of
hexosamine biosynthesis UDP-N-acetylglucosamine was higher
in the transgenic animals (P < 0.02) but was also
unaffected by troglitazone treatment.
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Figure 4. Hexosamine pathway activity in
troglitazone-treated animals. GFA activity (A) and
UDP-N-acetylglucosamine levels (B) in control and
transgenic mice untreated or treated with troglitazone.
Shown is mean ± SEM; n = 4/group.
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Discussion
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The hexosamine biosynthetic pathway has been hypothesized to be
involved in mediating some of the toxic effects of hyperglycemia (5, 22). Garvey et al. (23) showed that insulin-stimulated
glucose uptake in primary cultures of adipocytes could be inhibited by
treating the cells with 20 mM glucose for 24 h. They
further determined that the cause of this insulin resistance was an
inhibition of GLUT4 translocation from intracellular vesicles to the
plasma membrane, with no change in GLUT4 protein levels. Through a
series of experiments, Marshall et al. (5) subsequently
demonstrated that this glucose-induced inhibition of insulin-stimulated
glucose uptake was caused by increased flux of glucose into hexosamine
biosynthesis: glucose-induced reductions in glucose uptake could be
blocked by inhibitors of hexosamine synthesis, and this blockade could
be bypassed, at higher potency, by glucosamine, which enters the
hexosamine pathway after being phosphorylated by hexokinase to form
glucosamine-6-phosphate. Subsequently, infusion of glucosamine into
whole animals was found to cause a decrease in insulin’s ability to
stimulate glucose uptake in vivo (6, 7); in the latter
study, muscle GLUT4 translocation was inhibited by glucosamine, with no
change in GLUT4 protein levels (7). All of these studies have led to
the hypothesis that some of the adverse effects of hyperglycemia, and
perhaps the pathogenesis of type 2 diabetes itself, may be linked to
hexosamine biosynthesis. The current studies strengthen this link by
demonstrating the similarity of hexosamine-induced insulin resistance
to type 2 diabetes. In both conditions, skeletal muscle glucose uptake
is impaired, secondary to defective insulin-stimulated translocation of
GLUT4, and both conditions can be treated with agonists of PPAR,
such as troglitazone.
Early strategies to investigate the effects of increased hexosamine
synthesis and its effects on insulin-stimulated glucose disposal
in vivo have involved infusions with glucose or glucosamine.
Glucosamine affects other tissues involved in glucose homeostasis,
specifically impairing the suppression of HGO by hyperglycemia (24) and
impairing insulin secretion (25). Furthermore, at high concentrations,
glucosamine has effects not directly related to increases in hexosamine
flux. For example, glucosamine treatment can deplete cellular ATP (26)
and directly affect enzymes such as glucokinase (24). Thus, to avoid
the multiple effects of infusing animals with glucosamine and to
implicate the hexosamine pathway more directly in the regulation of
insulin-stimulated glucose uptake in skeletal muscle, our laboratory
has taken a tissue-specific transgenic approach to investigate the
effects of increased hexosamine biosynthesis. By overexpressing the
rate-limiting enzyme for hexosamine synthesis GFA in the target tissues
of insulin-stimulated glucose disposal, we sought to investigate the
effects of increased hexosamine biosynthesis in muscle and adipose
tissue, without the additional effects known to be caused by treatment
with nonphysiologic concentrations of glucosamine (24, 25, 26). With
approximately a 2-fold increase in skeletal muscle GFA activity in the
transgenic animals (9), total cellular glucose flux into the hexosamine
pathway would be estimated to increase from approximately 2% to only
4–6%, and thus should neither significantly alter glucose
availability for oxidative or nonoxidative metabolism nor cause
significant changes in cellular ATP pools.
Overexpression of GFA in skeletal muscle and adipose tissue of
transgenic mice did result in the inhibition of insulin-stimulated
glucose disposal in intact animals (9). These results are confirmed and
expanded in the current study, with the demonstration that the
transgenic mice are insulin resistant, compared with their
nontransgenic littermates, over a wide range of insulin doses, with no
difference in HGO. More importantly, we demonstrate directly that this
peripheral insulin resistance can be accounted for by a decrease in
glucose uptake, as determined by [3H]2-DOG uptake during
a hyperinsulinemic clamp.
The decrease in 2-DOG uptake in muscle overexpressing GFA is paralleled
by a significant defect in GLUT4 translocation after insulin
stimulation. Skeletal muscle from animals infused with glucosamine has
also been shown to be insulin resistant, primarily because of an
inhibition of GLUT4 translocation (7). Although we previously reported
a decrease in GLUT4 protein (but not messenger RNA) levels in these
transgenic mice (9), the current study demonstrates that skeletal
muscle GLUT4 protein levels, assessed by immunoblotting, did not differ
between the transgenic and control mice. The reasons for this
discrepancy are not clear. The previous immunoblots for GLUT4 were
performed using a different antibody and on mice whose genetic
background was in flux, because they were being back-bred onto a C57Bl6
background to facilitate the detection of diabetes- and obesity-related
phenotypes. The current study was performed using mice that had been
bred on the C57Bl6 background for more than 6 generations. Regardless
of the reasons for the discrepancy, the current evidence shows clearly
that the translocation defect can account for the defect in glucose
uptake. Thus, there seems to be a common mechanism by which diabetes,
glucosamine infusion, or overexpression of GFA cause insulin resistance
to glucose uptake in skeletal muscle.
Further evidence for the similarity of hexosamine-induced insulin
resistance to human type 2 diabetes is provided by reversal of the
insulin resistance in the transgenic animals by
troglitazone. The mechanism of action of PPAR agonists
is still unknown. Although the most parsimonious explanation of the
current results would involve a direct effect of the drug on skeletal
muscle, there is controversy about the possible contribution of muscle
to troglitazone action. Several recent studies have been
able to demonstrate PPAR messenger RNA and troglitazone
action in skeletal muscle and in muscle-derived cells (27, 28, 29, 30, 31).
Furthermore, PPAR levels in muscle have been reported to be altered
in insulin-resistant states (32), and there is rationale for PPAR
action in muscle, given the regulation by PPAR of genes involved in
fatty acid oxidation by muscle (33). At this time, however, we cannot
rule out an indirect effect of troglitazone, such as an
alteration in delivery of fuel or regulatory factors, from fat to
muscle, that reverses the hexosamine-induced defect.
The results on the effects of troglitazone are at variance
with those of a previous study wherein glucose-, but not
glucosamine-induced insulin resistance could be reversed by
troglitazone (34). However, the latter study employed an
acute infusion with a high concentration of glucosamine, and whether
this acute model is analogous to chronic diabetes or chronically
increased hexosamine flux is not known. In the absence of information
on the dose responsiveness of the insulin resistance for glucosamine or
troglitazone, the significance of the previously published
negative data is unclear. Furthermore, the appropriateness of using
acute glucosamine exposure to study hexosamine-induced insulin
resistance has been raised in a recent publication, indicating that
some of the observed effects on glucose uptake in glucosamine infused
animals might be accounted for by decreased ATP levels (26).
This was not the case in the current study, because the transgenic
animals did not exhibit decreased muscle ATP levels. This result was
not surprising, given the relatively small increase in glucose flux
into the hexosamine pathway achieved by overexpressing GFA. The results
do illustrate, however, the advantages of studying the effects of
hexosamine flux in the transgenic model system, as opposed to acute
exposure of cells and animals to high concentrations of
glucosamine.
The lack of an effect of troglitazone on GFA and
UDP-N-acetylglucosamine levels argues that the effect of
that drug in reversing insulin resistance is distal to the generation
of hexosamines. How increased hexosamine flux or
troglitazone exerts these effects on glucose uptake is
unknown. The final product of hexosamine synthesis,
UDP-N-acetylglucosamine, is a substrate for protein
glycosylation, both N- and O-linked. Recently,
O-linked glycosylation has been hypothesized to be a
regulatory modification, analogous to phosphorylation, that may play a
role in mediating the assembly of regulatory protein complexes (35).
Unlike N-linked glycosylation, O-linked
glycosylation is dynamic and has a rapid turnover rate. Identification
of the O-glycosylation sites on c-myc (36) and
RNA polymerase suggests a possible reciprocal functional relationship
of O-glycosylation with phosphorylation. Synthetic peptides
corresponding to sequences of glycogen synthase have been shown to be
effective substrates for O-glycosylation (35). Thus, both
transcriptional and posttranslational changes, mediated by
O-glycosylation, are candidate mechanisms by which cells use
hexosamine synthesis as a glucose-sensing mechanism (35, 36, 37).
In conclusion, we have demonstrated that overexpression of GFA, the
rate-limiting enzyme in hexosamine synthesis, in muscle and adipose
tissue of transgenic mice, causes insulin resistance over a wide range
of insulin doses. Furthermore, this insulin resistance is directly
demonstrated to be caused by a decrease in glucose uptake in skeletal
muscle, secondary to an inhibition of GLUT4 translocation from
intracellular vesicles to the plasma membrane. The thiazolidinedione
troglitazone reverses this insulin resistance by acting at
a point distal to the generation of hexosamines. Thus, the similarities
revealed between type 2 diabetes and hexosamine-induced insulin
resistance are striking. We have hypothesized that the hexosamine
pathway serves the as a satiety sensor for insulin-dependent tissues
(22); and these studies lend further support to the hypothesis that
increased glucose flux through the hexosamine pathway mediates adverse
effects of hyperglycemia, as well. Further characterization of these
mice should be instrumental in clarifying the role that hexosamines and
hexosamine synthesis play in cellular sensing of glucose and the
responses to hyperglycemia.
|
Acknowledgments
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|---|
We wish to thank Dr. Alain Baron (Indiana University) for help
in establishing the euglycemic clamp technique in mice.
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Footnotes
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1 This work was supported by the Research Services of the Department of
Veterans Affairs Medical Centers, Jackson, MS (to D.A.M.) and
Charleston, SC (to W.T.G.) and the National Institute of Health
[Grants DK-43526 (to D.A.M.) and DK-38765 (to W.T.G.)]. 
2 Both authors should be considered as first authors of this
manuscript.
Received September 18, 1998.
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Abstract
Introduction
