From the Department of Cell Biology and Physiology, Washington
University School of Medicine, St. Louis, Missouri 63110 and the
§ Diabetes Research Center, Vrije Universiteit Brussel,
B-1090, Brussels, Belgium
Glucosamine, which enters the hexosamine pathway
downstream of the rate-limiting step, has been routinely used to mimic
the insulin resistance caused by high glucose and insulin. We
investigated the effect of glucosamine on insulin-stimulated
glucose transport in 3T3-L1 adipocytes. The
-insulin
(insulin-stimulated minus basal) value for 2-deoxyglucose uptake was
dramatically inhibited with increasing concentrations of
glucosamine with an ED50 of 1.95 mM.
Subcellular fractionation experiments demonstrated that reduction in
insulin-stimulated 2-deoxyglucose uptake by glucosamine was due to an
inhibition of translocation of both Glut 1 and Glut 4 from the low
density microsomes (LDM) to the plasma membrane. Analysis of the
insulin signaling cascade revealed that glucosamine impaired insulin
receptor autophosphorylation, insulin receptor substrate (IRS-1)
phosphorylation, IRS-1-associated PI 3-kinase activity in the LDM, and
AKT-1 activation by insulin. Measurement of intracellular ATP
demonstrated that the effects of glucosamine were highly correlated
with its ability to reduce ATP levels. Reduction of intracellular ATP
using azide inhibited Glut 1 and Glut 4 translocation from the LDM to
the plasma membrane, insulin receptor autophosphorylation, and IRS-1
tyrosine phosphorylation. Additionally, both the reduction in
intracellular ATP and the effects on insulin action caused by
glucosamine could be prevented by the addition of inosine, which served
as an alternative energy source in the medium. We conclude that direct
administration of glucosamine can rapidly lower cellular ATP levels and
affect insulin action in fat cells by mechanisms independent of
increased intracellular UDP-N-acetylhexosamines and that
increased metabolism of glucose via the hexosamine pathway may not
represent the mechanism of glucose toxicity in fat cells.
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INTRODUCTION |
Insulin resistance is a major contributing factor in the
pathogenesis of long term complications of
non-insulin-dependent diabetes mellitus (1). Hyperglycemia
is known to cause insulin resistance. High concentrations of glucose
and insulin have been shown to impair insulin-stimulated glucose
transport in primary rat adipocytes (2, 3) and rat hind limb muscles
(4). Previous studies have suggested that an increased flux through the
hexosamine biosynthetic pathway may be the mechanism by which hyperglycemia leads to insulin resistance (5, 6). Only 2-3% of the
total glucose taken up by the cell is metabolized by this pathway that
ultimately produces UDP-N-acetylglucosamine, which serves as
a substrate in the formation of glycoproteins, glycolipids, and
proteoglycans. Three lines of evidence led Marshall et al. (5) to conclude that hexosamine biosynthesis is involved in insulin
resistance. First, glucose and insulin per se were not sufficient to establish insulin resistance of glucose transport in
primary adipocytes, but the presence of glutamine was essential (7, 8).
Second, desensitization could be prevented by inhibitors of the
rate-limiting enzyme of the pathway, glutamine-fructose-6-phosphate aminotransferase (GFAT)1 (5).
Third, the strongest evidence was that glucosamine, entering the
pathway downstream of the rate-limiting step, was 40 times more
effective than glucose in mediating desensitization of glucose transport (5). The effects of glucosamine were confirmed by many other
studies, in vitro, including studies in primary rat adipocytes (9), 3T3-L1 adipocytes (10), and isolated skeletal muscle
(11) and in vivo by measuring whole body glucose disposal after glucosamine clamping (12-14).
More direct evidence that the hexosamine pathway is involved in insulin
resistance is through the overexpression of GFAT. Insulin sensitivity
of glycogen synthase was decreased in fibroblasts stably transfected
with GFAT (15-17). Glucose disposal was decreased by half (18) in
transgenic mice overexpressing GFAT specifically in muscle and fat. In
addition, mice that overexpress Glut 1 in muscle were insulin-resistant
(19, 20) and had elevated GFAT expression compared with wild-type mice
or insulin-sensitive Glut 4 overexpressors (21). Interestingly,
however, a recent study that examined the effect of GFAT overexpression
on insulin-stimulated translocation of Glut 4 by co-transfecting
primary rat adipose cells with GFAT and an epitope-tagged Glut 4 transporter found that overexpression of GFAT had no effect on the
insulin dose-response curve for Glut 4 translocation compared with
control cells transfected with only tagged Glut 4 (9). The authors did
observe an inhibition of Glut 4 translocation when adipose cells were
treated with glucosamine.
Glucosamine has become routinely used to mimic the insulin resistance
caused by high glucose and insulin. It is naturally assumed that higher
intracellular concentrations of glucosamine will have the same
metabolic effect as increasing the flux through the hexosamine
biosynthetic pathway. It is not clear, however, what cellular process
is specifically affected by glucosamine to confer insulin resistance
and whether it is by the same mechanism as high glucose and insulin or
even the overexpression of GFAT. To address this question, we
investigated the effects of glucosamine on insulin-stimulated glucose
transport in 3T3-L1 adipocytes. Our results revealed that direct
administration of glucosamine can rapidly lower intracellular ATP
levels and affect insulin action in fat cells by mechanisms independent
of increased intracellular UDP-N-acetylhexosamines.
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EXPERIMENTAL PROCEDURES |
Treatment of 3T3-L1 Adipocytes--
3T3-L1 fibroblasts were
grown to confluence and 48 h later subjected to differentiation as
described previously (22). 3T3-L1 adipocytes were used 10-14 days
after differentiation. Cells treated with glucosamine were first washed
three times with PBS and then incubated for 1 h at 37 °C in
glucose-free DMEM supplemented with 1 mM sodium pyruvate, 6 nM insulin, and various concentrations of glucosamine
(Sigma). L-Glucose was added to adjust the osmolarity of
the sugars to 25 mM. Following three washes with PBS, cells were incubated for 1.5 h in the same media but without insulin. Control cells were also pretreated with insulin in the same manner but
were incubated in either normal DMEM (25 mM
D-glucose) or in glucose-free DMEM supplemented with 25 mM L-glucose. Media containing more than 5 mM glucosamine were adjusted to pH 7.4.
Cells used in the sodium azide studies were fed with DMEM supplemented
with 10% fetal bovine serum the day prior to the experiment. Fully
differentiated 3T3-L1 adipocytes were washed three times with PBS and
then incubated for 2.5 h at 37 °C in glucose-free DMEM
containing 6 or 7 mM sodium azide.
2-Deoxyglucose Uptake
Measurements--
[3H]2-Deoxyglucose uptake was measured
as described previously (22). After the 2.5-h preincubation described
above, cells were washed three times with Krebs-Ringer phosphate and
then treated or not treated for 20 min at 37 °C with 1 µM insulin. Non-transport-mediated uptake was measured in
the presence of 20 µM cytochalasin B. Protein content was
measured using the bicinchoninic acid method (Pierce).
Subcellular Fractionation--
Three culture dishes (10 cm) of
3T3-L1 adipocytes were pretreated as described above, incubated for 20 min at 37 °C with or without 1 µM insulin, and then
washed three times with ice-cold PBS containing 1 mM sodium
vanadate. Cells were scraped in 12 ml of ice-cold HES buffer (255 mM sucrose, 20 mM HEPES, pH 7.4, and 1 mM EDTA) supplemented with 100 mM sodium
fluoride, 10 mM sodium pyrophosphate, 1 mM
sodium vanadate, and protease inhibitors and then homogenized by
passing the cells 11 times through a Yamato LSC homogenizer at a speed
of 1200 rotations/min at 4 °C. Subcellular fractionation was carried
out by differential centrifugation as described previously (23). The
following protease inhibitors were used: 1 µg/ml leupeptin, 1 µg/ml
antipain, 1 µg/ml benzamidine, 5 µg/ml trypsin inhibitor, 1 µg/ml
chymostatin, 1 µg/ml pepstatin A, and 0.5 mM
phenylmethylsulfonyl fluoride.
Western Blot Analysis--
50 µg of protein were subjected to
SDS-PAGE and then transferred to nitrocellulose. Glut 1 and Glut 4 transporters were detected by Western blot analysis using specific
polyclonal antibodies generated against the final 16 amino acids of
each glucose transporter. Phosphotyrosine-phosphorylated proteins were
detected using the monoclonal PY-20 antibody (Transduction
Laboratories). 125I-Labeled goat anti-rabbit or anti-mouse
IgG (0.25 µCi/ml, ICN, Irvine, CA) was used as the secondary
antibody. Radioactive bands were quantitated by a PhosphorImager SI
Analyzer (Molecular Dynamics).
PI 3-Kinase Activity--
Cells were treated with 25 mM D-glucose or 20 mM glucosamine
for 2.5 h, in which the first hour was in the presence of 6 nM insulin. The cells were then washed and incubated in the
same media without insulin for the remaining 1.5 h. Cells were
then fractionated after 0, 2, 5, and 15 min of acute 1 µM
insulin stimulation as described above. 100 µg of LDM protein were
solubilized with 1 ml of 20 mM Tris-HCl, 137 mM
NaCl, 1 mM MgCl2, 1 mM
CaCl2, 1 mM sodium vanadate, 100 mM
NaF, 10 mM sodium pyrophosphate, 1% Nonidet P-40, and
protease inhibitors and then immunoprecipitated with 10 µl of a
polyclonal antibody directed against the carboxyl-terminal 14 amino
acids of rat liver IRS-1. Immune complexes were bound to protein
A-Sepharose beads, and PI 3-kinase activity was determined on the
pellets as described by Backer et al. (24). The resulting 32P-labeled phosphatidylinositol 3-phosphate lipids were
separated from the other reaction products by thin layer chromatography (24). Autoradiograms were then quantified by PhosphorImager analysis.
Insulin Receptor Purification and in Vitro
Phosphorylation--
Two 150-cm culture plates of 3T3-L1 adipocytes
were treated with 25 mM D-glucose, 20 mM L-glucose, or 20 mM glucosamine
for 2.5 h, in which the first hour was carried out in the presence of 6 nM insulin and the remaining 1.5 h was without
insulin. Cells were washed three times and then scraped in 12 ml of
ice-cold buffer (50 mM HEPES, pH 7.5, 1 mM
EDTA) supplemented with protease inhibitors. Cells were homogenized by
passing them 11 times through a Yamato LSC homogenizer at a speed of
1200 rotations/min at 4 °C, total membranes were prepared, and
insulin receptors were semipurified by wheat germ agglutinin-Sepharose
as described previously (25). 1 µM insulin was added or
not added for 20 min to an aliquot of insulin receptor (10 µg of
total protein in 0.1% Triton X-100). Receptors were autophosphorylated
for 5 min at room temperature in 50 mM HEPES (pH 6.9),
0.1% Triton X-100, 5 mM manganous acetate, and 20 µM [
-32P]ATP (25-50 cpm/fmol).
RCM-lysozyme (Sigma), was added for 1 min at a final concentration of
100 µM before the reactions were quenched with the
addition of EDTA (20 mM final) and analyzed by
SDS-PAGE.
ATP Analysis--
3T3-L1 adipocytes were pretreated as described
above. Culture plates (3.5 cm) were washed four times with ice-cold PBS
and then solubilized with 0.4 ml of ice-cold 0.05 N NaOH.
The cell extracts were sheared using a 25-gauge syringe. 120 µl were
heated to 80 °C for 20 min and then neutralized with 60 µl of 0.1 M Tris-HCl, pH 6.8, 0.05 N HCl. Protein content
was measured and ATP concentrations were determined using the method of
Passonneau and Lowry (26). In this procedure, NADPH is assayed as a
measure of ATP by using a two-step coupled reaction in which in the
first step glucose and hexokinase are added to the ATP sample to
produce ADP and glucose 6-phosphate. In the second step, glucose
6-phosphate is converted to NADPH and 6-phosphogluconolactone by NADP
and glucose-6-phosphate dehydrogenase. NADPH is then measured
fluorometrically. To 20 µl of the neutralized cell extract solution,
100 µl of 50 mM Tris-HCl, pH 8.1, 0.02% bovine serum
albumin, 1 mM MgCl2, 0.5 mM
dithiothreitol, 100 µM glucose, 100 µM
NADP+, 2 µg/ml hexokinase, and 0.5 µg/ml leuconostoc
mesenteroides glucose-6-phosphate dehydrogenase (Calbiochem) were added
and incubated for 20 min at room temperature. Excess NADP+
was then destroyed with the addition of 10 µl of 0.7 N
NaOH at 60 °C for 20 min. The fluorescence signal of NADPH was
enhanced 8-fold by the addition of 1 ml of 6 N NaOH, 10 mM imidazole base, and 0.01% H2O2
(26). Samples were mixed immediately upon addition and incubated for 20 min at 60 °C, and then the fluorescence was measured at 340 nm
excitation and 460 nm emission. The fluorescence intensity was
converted to µmol/liter using an ATP standard curve generated under
identical conditions. Duplicates were done for each sample, blank, and
standard curve measurement. ATP values were corrected for protein
content in the sample and then normalized to control ATP values
(glucose-free DMEM).
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RESULTS |
Effect of Glucosamine on Glucose Transport in 3T3-L1
Adipocytes--
To determine the effects of increasing flux through
the hexosamine pathway on glucose transport, 3T3-L1 adipocytes were
incubated with increasing concentrations of glucosamine in glucose-free DMEM for 1 h in the presence of 6 nM insulin. The
cells were then washed, incubated in the same media without insulin for
1.5 h, then acutely stimulated or not stimulated with 1 µM insulin prior to measuring 2-deoxyglucose uptake (Fig.
1A). Glucose-free media were
used, since glucosamine competes poorly with glucose for transport into
cells. The 1-h incubation with insulin was done to increase the number
of glucose transporters on the plasma membrane and thus facilitate
glucosamine uptake. Insulin was removed after 1 h to restore acute
insulin sensitivity. In the absence of glucose and glucosamine under
these experimental conditions, 2-deoxyglucose uptake increased
2.75-fold with acute insulin from 0.363 to 1.0 on a normalized scale.
The rather low -fold increase in insulin-stimulated uptake is due to a
glucose starvation effect. Incubation of 3T3-L1 adipocytes in
glucose-free DMEM results in a higher basal and insulin-stimulated
2-DOG uptake, which is caused largely by an increase in the number of
Glut 1 transporters at the plasma membrane (27). When 2-deoxyglucose
uptake experiments were performed under identical conditions as
described above but in the presence of 25 mM
D-glucose and no glucosamine, uptake increased from
0.06 ± .008 to 0.77 ± 0.027, which is a 12.8-fold increase
(data not shown). If cells are treated with increasing concentrations
of glucosamine (glucose-free DMEM), 2-DOG uptake in the presence of
insulin decreased dramatically (Fig. 1A). Basal uptake also decreased but at higher glucosamine concentrations (>5
mM). The change in 2-DOG uptake with insulin (
-insulin)
was plotted as a function of glucosamine concentration (Fig.
1B). The ED50 was 1.95 mM, which is
between the values reported by Marshall et al. (5) for
glucosamine treatment (5 h) of primary rat adipocytes performed in the
absence (ED50 = 6.7 mM) and presence of insulin (ED50 = 0.36 mM).

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Fig. 1.
Effect of glucosamine on glucose transport in
3T3-L1 adipocytes. A, 3T3-L1 adipocytes were incubated
for 1 h in the presence of 6 nM insulin in
glucose-free DMEM containing 0, 0.5, 1, 2, 5, 10, or 20 mM
glucosamine, adjusted to equal osmolarity with L-glucose.
Following three washes with PBS, cells were incubated in the same media
without insulin for 1.5 h. [3H]2-Deoxyglucose uptake
was measured for 6 min under basal ( ) or acute insulin stimulation
(20 min at 37 °C) with 1 µM insulin ( ). Uptake was
quantitated as pmol of [3H]2-deoxyglucose/min/mg of
cellular protein normalized to insulin-stimulated control cells
(glucose-free DMEM, 0 mM glucosamine). The actual value of
2-DOG uptake for insulin-stimulated glucose-free control cells was
794 ± 13.97 pmol of [3H]2-DOG/min/mg of protein.
Data represent the mean ± S.E. of at least three independent
experiments. S.E. values not shown are hidden by the
symbols. B, -insulin values
(insulin-stimulated minus basal 2-DOG uptake) were calculated for each
experiment followed by mean and S.E. determination.
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Effect of Glucosamine on Glut 1 and Glut 4 Translocation--
Glut
1 and Glut 4 transporters have been shown to translocate to the plasma
membrane (PM) with insulin from a low density microsomal fraction (LDM)
in both primary adipose cells (28) and 3T3-L1 adipocytes (23).
Subcellular fractionation of 3T3-L1 adipocytes was carried out (see
"Experimental Procedures") after treating cells with
D-glucose-free DMEM containing L-glucose alone or in the presence of 2, 5, and 20 mM glucosamine. The
amounts of Glut 1 (Fig. 2A)
and Glut 4 (Fig. 2B) in the plasma membrane for both basal
and insulin-treated cells were determined by Western blot analysis. For
control cells incubated in L-glucose DMEM (no glucosamine),
the amount of Glut 1 transporter at the PM tended to increase with
insulin (from 0.7 to 1.0) although it was not statistically
significant. If cells were incubated in DMEM containing 25 mM D-glucose, Glut 1 content in the PM
increased from 0.47 to 1.08 with insulin (data not shown). The observed
increase in Glut 1 at the PM in the basal state under glucose
starvation conditions is consistent with previously published data (27)
and is also in agreement with the 2-DOG uptake results described above.
Glut 4 increased in the PM approximately 2-fold (from ~0.5 to 1.0) with insulin in cells incubated with glucosamine-free DMEM containing either L-glucose (Fig. 2B) or
D-glucose (data not shown). Glucosamine decreased
insulin-stimulated translocation of both Glut 1 and Glut 4 to the
plasma membrane. At 2 mM glucosamine, translocation of Glut
1 and Glut 4 with insulin was inhibited 80 and 70%, respectively, and
translocation of both transporters was completely abolished at 20 mM glucosamine. The severity of the inhibition especially at 2 mM glucosamine was higher than one would have
predicted based on the
-insulin uptake results (ED50 = 1.95 mM). The apparent inconsistency is probably due to the
fact that the PM fractions were contaminated with other subcellular
compartments that also contain each of the transporters, which masked
the degree of insulin-activated translocation to the PM. Therefore, any
translocation that we did observe in the presence of glucosamine was
not statistically significant.

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Fig. 2.
The effect of glucosamine on the
translocation of Glut 1 (G1) and Glut 4 (G4)
from the low density microsomes to the plasma membrane. 3T3-L1
adipocytes were incubated for 1 h in the presence of 6 nM insulin in glucose-free DMEM containing 0, 2, 5, 10, or
20 mM glucosamine, adjusted to equal osmolarity with
L-glucose. Following three washes with PBS, cells were
incubated in the same media without insulin for 1.5 h. Cells were
then treated (hatched bars) or not treated
(black bars) with 1 µM insulin for
20 min, washed with ice-cold buffer containing phosphatase and protease
inhibitors, and then homogenized. Subcellular fractionation was carried
out by differential centrifugation (see "Experimental Procedures").
PM and LDM fractions were separated on SDS-PAGE (50 µg of protein),
immunoblotted, and incubated with antibodies raised against the
carboxyl terminus of either Glut 1 or Glut 4. Signal intensities from
125I-labeled secondary antibodies were quantitated by
PhosphorImager analysis. Data represent the mean ± S.E. of three
independent experiments. #, relative abundance differed significantly
(p < 0.05) in the stimulated state compared with the
basal state. *, relative abundance in the basal glucosamine samples
differed significantly compared with the basal control samples. An
unpaired Student's t test was used in all statistical
analyses. A, amount of Glut 1 transporter at the PM plotted
relative to insulin-stimulated control cells. B, amount of
Glut 4 transporter at the PM plotted relative to insulin-stimulated
control cells. C, amount of Glut 1 transporter in the LDM
plotted relative to basal control cells. D, amount of Glut 4 transporter in the LDM plotted relative to basal control cells.
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Insulin-stimulated depletion of Glut 1 and Glut 4 from the LDM is shown
in Fig. 2, C and D, respectively. In control
cells (L-glucose DMEM) the amount of Glut 1 and Glut 4 in
the LDM decreased with insulin from 1.0 to 0.43 and from 1.0 to 0.67, respectively. Glucosamine inhibited insulin-induced translocation from
the LDM for both Glut 1 and Glut 4 in a
concentration-dependent manner. At 2 mM
glucosamine, only Glut 1 was statistically depleted from the LDM with
insulin treatment. Interestingly, the absolute amount of Glut 4 in the
LDM under basal conditions was significantly higher with glucosamine
treatment compared with the control (L-glucose), suggesting
that glucosamine caused a redistribution of Glut 4 to the LDM under
basal conditions. A similar glucosamine-induced redistribution of Glut
4 to the LDM in the basal state was observed in primary rat adipocytes
(29).
Effect of Glucosamine on Insulin Signaling--
The insulin
signaling pathway was investigated to determine the mechanism by which
glucosamine inhibits insulin-stimulated glucose transporter
translocation. Although the complete insulin pathway for translocation
is still unclear, several of the initial steps have been identified in
recent years (30). The cascade is initiated by binding of insulin to
specific cell surface receptors that then autophosphorylate critical
tyrosine residues that activate an intrinsic tyrosine kinase. In fat
cells, insulin-stimulated tyrosine phosphorylation of IRS-1 has been
shown to be important in the translocation of Glut 4 (31). Transmission
of the insulin signal from the cell surface to the LDM appears to
involve phosphorylation of IRS-1 by the insulin receptor at the PM (32)
and movement of tyrosine-phosphorylated IRS-1 to the LDM, where it
binds and activates PI 3-kinase to generate phosphatidylinositol
3,4,5-phosphate lipids (33). Phosphatidylinositol 3,4,5-phosphate
lipids bind to and activate protein kinase B (AKT) kinase (34) that
phosphorylates and activates AKT, which somehow causes vesicles
containing Glut 4 to migrate to the cell surface (35, 36). We first
investigated the autophosphorylation step of the insulin receptor by
performing Western blot analyses on PM fractions using an
anti-phosphotyrosine antibody (Fig. 3).
Insulin stimulated tyrosine autophosphorylation of the insulin receptor
20-fold in L-glucose control cells (Fig. 3A).
Glucosamine inhibited insulin-activated autophosphorylation dramatically in a concentration-dependent manner.
-Insulin autophosphorylation was reduced 52, 64, and 72% at 2, 5, and 20 mM glucosamine, respectively, compared with
L-glucose controls. LDM fractions were then analyzed for
tyrosine phosphorylation to examine the phosphorylation of IRS-1 by the
insulin receptor. Insulin stimulated IRS-1 tyrosine phosphorylation
approximately 12-fold in control cells (Fig. 3B). Glucosamine inhibited
-insulin tyrosine phosphorylation of IRS-1 to
approximately 50% of that observed in L-glucose controls
when cells were treated with 2, 5, or 20 mM glucosamine. On
the whole, insulin receptor autophosphorylation was more sensitive to
glucosamine treatment than IRS-1 tyrosine phosphorylation. This is
consistent with the idea of a spare insulin receptor population.

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Fig. 3.
The effect of glucosamine on insulin receptor
autophosphorylation and IRS-1 phosphorylation. 3T3-L1 adipocytes
were incubated in glucose-free DMEM containing 0, 2, 5, 10, or 20 mM glucosamine for 2.5 h and then fractionated as
described under "Experimental Procedures." Cells were then treated
(hatched bars) or not treated (black
bars) with 1 µM insulin for 20 min. 50 µg of
PM fractions (A) and 50 µg of LDM (B) were
separated by SDS-PAGE, immunoblotted, and probed with an antibody
against phosphotyrosine (PY-20). Signal intensities from
125I-labeled secondary antibodies were quantitated by
PhosphorImager analysis. Data represent the mean ± S.E. of three
independent experiments. #, relative abundance differed significantly
(p < 0.05) in the stimulated state compared with the
basal state. *, relative abundance in the insulin-stimulated
glucosamine samples differed significantly compared with the stimulated
control samples.
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The next step in the cascade, IRS-1-associated PI 3-kinase activity,
was analyzed using LDM fractions from control and glucosamine-treated cells. LDM were solubilized with Nonidet P-40 and then
immunoprecipitated with IRS-1 antibody. PI 3-kinase activity was
measured in the immune complexes bound to protein A-Sepharose beads as
described under "Experimental Procedures." The IRS-1-associated PI
3-kinase activity in control cells increased during the first 5 min
after insulin addition to 8.4-fold basal activity and then dropped to 5-fold (Fig. 4A). In
glucosamine cells, the IRS-1-complexed PI 3-kinase activity was 30% of
control after 2 min of insulin, 35% after 5 min of insulin, and 50%
after 15 min of insulin.

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Fig. 4.
The effect of glucosamine on IRS-1-associated
phosphatidylinositol 3-kinase activity in LDM and AKT activation.
A, cells were treated with 25 mM
D-glucose ( ) or 20 mM glucosamine ( ) for
2.5 h using the protocol under "Experimental Procedures."
Cells were then fractionated after 0, 2, 5, and 15 min of acute insulin
stimulation. 100 µg of LDM protein were solubilized with Nonidet P-40
and then immunoprecipitated with IRS-1 antibody. Immune complexes were
bound to protein A-Sepharose beads, and PI 3-kinase activity was
determined on the pellets as described under "Experimental
Procedures." The resulting phosphatidylinositol 3-phosphate lipids
were separated from the other reaction products by thin layer
chromatography. Autoradiograms were then quantified by PhosphorImager
analysis. Data were expressed as relative abundance versus
basal control (D-glucose) and represent the average of ± S.E. of three independent experiments. B, cells were
treated with 25 mM D-glucose
(CONTROL) or 20 mM glucosamine (GLN)
using the protocol under "Experimental Procedures." After 20 min of
acute insulin activation or no activation, cells were homogenized and
fractionated. 50 µg of cytosol were separated by SDS-PAGE,
immunoblotted, probed with an antibody against AKT-1 (Upstate
Biotechnology Inc.), and visualized by ECL.
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AKT activation by insulin was analyzed from AKT-1 immunoblots of
cytosol from control and glucosamine-treated cells. Insulin causes an
increase in serine and threonine phosphorylation of AKT, which shifts
the migration of the protein to a higher molecular mass on a
SDS-polyacrylamide gel (37). This shift was observed using control LDM
but was absent with LDM from glucosamine-treated cells (Fig.
4B).
The most likely interpretation of our signaling data is that
glucosamine impaired insulin-stimulated translocation by inhibiting insulin receptor autophosphorylation, which would then progressively affect each subsequent step in the cascade. To determine whether glucosamine treatment modified the receptor directly, we performed in vitro phosphorylation reactions using semipurified
insulin receptors isolated from glucosamine-treated and control cells. Cells were incubated in DMEM containing D-glucose,
L-glucose, or 20 mM glucosamine for 2.5 h
using our standard protocol. Insulin receptors were then semipurified
from isolated membranes using wheat agglutinin-Sepharose. In
vitro phosphorylation of RCM lysozyme (38), a synthetic substrate
of the insulin receptor tyrosine kinase, was carried out with receptors
that were first preactivated or not preactivated with 1 µM insulin for 20 min and then autophosphorylated for 5 min with [32P]ATP. After 1 min of substrate
phosphorylation, the reactions were quenched and analyzed by SDS-PAGE.
The results, shown in Fig. 5, indicate
that insulin stimulated autophosphorylation of the insulin receptor
-subunit approximately 2-fold and phosphorylation of RCM-lysozyme
about 5-fold regardless of whether the receptors were prepared from
control or glucosamine-treated cells. These results demonstrate that
the intrinsic tyrosine kinase activity of the insulin receptor was not
directly affected by glucosamine treatment.

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Fig. 5.
In vitro tyrosine kinase activity of
insulin receptors prepared from control and glucosamine-treated
cells. Cells were treated with 25 mM
D-glucose, 20 mM L-glucose, or 20 mM glucosamine for 2.5 h in which the first hour was
carried out in the presence of 6 nM insulin and the
remaining 1.5 h was without insulin. Insulin receptors were
semipurified by wheat germ agglutinin-Sepharose using total membranes
prepared from these cells as described under "Experimental
Procedures." 1 µM insulin was added or not added
in vitro for 20 min to an aliquot of the insulin receptor.
Autophosphorylation was initiated with the addition of 50 mM HEPES (pH 6.9), 5 mM manganous acetate, and
20 µM [ -32P]ATP (all final
concentrations). After 5 min, RCM-lysozyme (100 µM final
concentration) was added. Reactions were quenched 1 min later by the
addition of EDTA (20 mM final concentration) and then
analyzed by SDS-PAGE.
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Effect of Glucosamine on Intracellular ATP Levels in 3T3-L1
Adipocytes--
Since the insulin receptor itself was not directly
altered by glucosamine treatment, the observed decrease in in
vivo insulin receptor autophosphorylation and substrate
phosphorylation may be caused by a reduction in intracellular ATP
levels by glucosamine. Glucosamine is taken up and is rapidly
phosphorylated by hexokinase at the expense of ATP. Since glucose was
absent from the media, only the exogeneously added pyruvate or the
endogeneous proteins, polysaccharides, or fat could be utilized to
replenish ATP. Therefore, if ATP were used faster than it was made, ATP
levels would fall. The effect of increasing concentrations of
glucosamine on intracellular ATP levels is shown in Fig.
6A. The ATP concentration of
cells incubated in glucose-free DMEM (5.17 mM ± 0.18) was
normalized to 1.0. Interestingly, the ATP concentration in cells
incubated in 25 mM D-glucose was slightly lower
(4.86 ± 0.15 mM). Therefore, in the absence of
glucosamine, glucose-starved cells can adequately maintain energy
levels. However, intracellular ATP levels rapidly decreased with
increasing concentrations of glucosamine (ED50 = 1.3 mM). The decline in ATP values closely followed the
decrease in
-insulin 2-DOG uptake (Fig. 1B). This is more
dramatically illustrated in Fig. 6B as a plot of relative
ATP concentration versus
-insulin 2-DOG uptake
(r = 0.934). To rule out the possibility that
glucosamine or glucosamine 6-phosphate in the cellular extracts interfered with the ATP analysis, we assayed extracts from
L-glucose control cells or cells treated with either 2 or
20 mM glucosamine in which we added a known quantity of
ATP. The amount of ATP we added to the extracts would have in theory
increased the intracellular ATP concentration by 3.5 mM.
The actual concentrations of additional ATP we measured in the control,
2 mM glucosamine-treated, and 20 mM
glucosamine-treated extracts were 3.49 ± 0.05, 3.52 ± 0.02, and 3.32 ± 0.02 mM, respectively. Since we could,
respectively, recover 99.7, 100.6, and 94.6% of the ATP we added to
the extracts, we conclude that nothing in the glucosamine extracts
interfered with the ATP assay.

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Fig. 6.
Effect of glucosamine on intracellular ATP
levels in 3T3-L1 adipocytes. A, 3T3-L1 adipocytes were
incubated for 2.5 h with glucose-free DMEM containing 0, 0.5, 1, 2, 5, 10, or 20 mM glucosamine as described under
"Experimental Procedures." Cells were then washed three times with
ice-cold PBS and then solubilized with 0.5 M NaOH. ATP
concentrations were determined as described under "Experimental
Procedures." Data represent the mean ± S.E. of at least three
independent experiments and are expressed relative to control cells
(L-glucose DMEM, 0 mM glucosamine). The actual
intracellular ATP concentrations of cells incubated in
L-glucose DMEM and 25 mM D-glucose
DMEM were 5.17 ± 0.18 and 4.86 ± 0.15 mM,
respectively. B, relative ATP values in A are
plotted versus -insulin 2-DOG uptake values shown in Fig.
1B. r represents the correlation coefficient of a
linear regression.
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Effect of Sodium Azide on Glucose Transporter Translocation and
Insulin Signaling--
It is possible that in our system glucosamine
may inhibit insulin action in ways other than by lowering ATP
concentrations and that the effect on ATP may not be the principal
cause of the insulin resistance. To strengthen the correlation between
decreases in intracellular ATP levels and effects on glucose
transporter translocation and insulin signaling, 3T3-L1 adipocytes were
treated with sodium azide for 2.5 h in order to lower ATP levels
by an independent mechanism (inhibition of mitochondrial electron
transport) and determine whether decreased energy levels per
se could produce the same effects seen with glucosamine treatment.
The ability of azide to reduce intracellular ATP levels is shown in
Fig. 8C. 6 and 7 mM azide for 2.5 h
decreased cellular ATP to 0.35 and 0.18, respectively, compared with
control cells (L-glucose DMEM), which is equivalent to
treating cells with approximately 3.5 and 20 mM
glucosamine, respectively (see Fig. 6A). Insulin-stimulated translocation of both Glut 1 and Glut 4 to the plasma membrane was
reduced with azide in a concentration-dependent manner as shown in Fig. 7, A and
B, respectively. In the basal state, the amount of Glut 1 and Glut 4 on the cell surface was largely unaffected by azide except
for a slight but not statistically significant reduction of Glut 4 with
7 mM azide. Similarly, translocation from the LDM with
insulin of Glut 1 (Fig. 7C) and Glut 4 (Fig. 7D)
was also decreased with increasing azide. Effects on insulin signaling
are shown in Fig. 8. Insulin-stimulated
tyrosine autophosphorylation of the insulin receptor at the plasma
membrane was dramatically inhibited with increasing azide
concentrations (Fig. 8A); however, tyrosine phosphorylation
of IRS-1 in the LDM by insulin was reduced only at the highest azide
concentration of 7 mM (Fig. 8B). This same
differential reduction in tyrosine phosphorylation between the insulin
receptor and IRS-1 was also observed with glucosamine treatment (Fig.
3).

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Fig. 7.
Effect of azide on Glut 1 (G1)
and Glut 4 (G4) translocation in 3T3-L1 adipocytes.
3T3-L1 adipocytes were treated for 2.5 h in glucose-free DMEM
containing 0, 6, or 7 mM sodium azide. Cells were treated
(hatched bars) or not treated (black
bars) with 1 µM insulin for 20 min prior to
subcellular fractionation (see "Experimental Procedures"). PM and
LDM fractions were separated on SDS-PAGE (50 µg of protein),
immunoblotted, and incubated with antibodies raised against the
carboxyl terminus either of Glut 1 or Glut 4. Signal intensities from
125I-labeled secondary antibodies were quantitated by
PhosphorImager analysis. Data represent the mean ± S.E. of three
independent experiments. #, relative abundance differed significantly
(p < 0.05) in the stimulated state compared with the
basal state. *, relative abundance in the insulin-stimulated azide
samples differed significantly compared with the stimulated control
samples. A, amount of Glut 1 transporter at the PM plotted
relative to insulin-stimulated control cells. B, amount of
Glut 4 transporter at the PM plotted relative to insulin-stimulated
control cells. C, amount of Glut 1 transporter in the LDM
plotted relative to basal control cells. D, amount of Glut 4 transporter in the LDM plotted relative to basal control cells.
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Fig. 8.
Effect of azide on insulin signaling in
3T3-L1 adipocytes. 3T3-L1 adipocytes were treated for 2.5 h
in glucose-free DMEM containing 0, 6, or 7 mM sodium azide.
Cells were then treated (hatched bars) or not
treated (black bars) with 1 µM
insulin for 20 min prior to subcellular fractionation (see
"Experimental Procedures"). PM and LDM fractions were separated on
SDS-PAGE (50 µg of protein), immunoblotted, and incubated with an
anti-phosphotyrosine-specific antibody (PY-20). Signal intensities from
125I-labeled secondary antibodies were quantitated by
PhosphorImager analysis. Data represent the mean ± S.E. of three
independent experiments. *, relative abundance in the
insulin-stimulated azide samples differed significantly
(p < 0.05) compared with insulin-stimulated control
samples. #, relative abundance differed significantly in the stimulated
state compared with the basal state. A, amount of
tyrosine-phosphorylated insulin receptor at the PM plotted relative to
insulin-stimulated control cells. B, the amount of
tyrosine-phosphorylated IRS-1 in the LDM plotted relative to
insulin-stimulated control cells. C, ATP concentrations were
determined as described (see "Experimental Procedures") and plotted
relative to control values. *, ATP concentrations in the azide-treated
cells were significantly different than that in control cells.
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Inosine Prevents Glucosamine-induced Effects in 3T3-L1
Adipocytes--
Next we attempted to prevent the glucosamine-induced
reduction in ATP levels by supplementing the media with an additional energy source to determine whether glucosamine can still affect insulin-stimulated glucose uptake and translocation without depleting ATP. Glucose was not used, since it competes very effectively for
uptake with glucosamine, and therefore a negative result would be more
difficult to interpret. Pyruvate and lactate (10 mM each) failed to prevent ATP declines with glucosamine (data not shown). Inosine has been shown to increase ATP concentrations in red blood cells (39), and at a concentration of 20 mM it did inhibit
the reduction in ATP with low glucosamine concentrations (<2
mM) in 3T3-L1 adipocytes (Fig.
9B). Inosine is taken up by
the cell and phosphorylated to IMP at the expense of one ATP. IMP is
broken down to hypoxanthine and ribose 5-phosphate. Ribose 5-phosphate is converted to 3-phosphoglyceraldehyde, which then enters the glycolytic pathway to generate pyruvate and two molecules of ATP. Although highly inefficient, each inosine molecule taken up generates one ATP molecule. Therefore, it was not surprising that inosine had
little effect on maintaining ATP levels at higher glucosamine concentrations (data not shown). The effect of inosine on preventing glucosamine-induced decreases in
-insulin 2-DOG uptake is shown in
Fig. 9A. The progressive decrease in
-insulin uptake by
low concentrations of glucosamine was eliminated with 20 mM
inosine. The uptake results correlated quite well with ATP values (Fig. 9B). Glucosamine decreased intracellular ATP levels in a
concentration-dependent manner, and 20 mM inosine prevented
the ATP reduction.

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Fig. 9.
Prevention of glucosamine-induced insulin
resistance of 2-deoxyglucose uptake by inosine. 3T3-L1 adipocytes
were incubated for 1 h in the presence of 6 nM insulin
in glucose-free DMEM containing 0, 0.5, 1, or 2 mM
glucosamine, adjusted to equal osmolarity with L-glucose.
In one set of culture plates nothing else was added (black
bars), and in the other set 20 mM inosine was
supplemented (hatched bars). Following three
washes with PBS, cells were incubated in the same media without insulin
for 1.5 h. A, [3H]2-deoxyglucose uptake
was measured for 6 min under basal or acute insulin stimulation (20 min
at 37 °C) with 1 µM insulin. Uptake was quantitated as
pmol of [3H]2-deoxyglucose/min/mg of cellular protein.
-insulin values were calculated and normalized to control cells (0 mM glucosamine with or without inosine). Data represent the
mean ± S.E. of at least three independent experiments. *,
relative -insulin 2-DOG uptake in the glucosamine cells differed
significantly (p < 0.05) from that in the control
cells. B, ATP concentrations were determined as described
(see "Experimental Procedures") and normalized to control values (0 mM glucosamine with or without inosine). Data represent the
mean ± S.E. of at least three independent experiments. *,
relative ATP concentrations in the glucosamine cells differed
significantly (p < 0.05) from that in the control
cells.
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Next we studied the effect of inosine on glucosamine-induced inhibition
of Glut 1 and Glut 4 translocation with insulin. 3T3-L1 adipocytes were
fractionated after treatment with D-glucose-free DMEM
containing L-glucose, 2 mM glucosamine, or 2 mM glucosamine supplemented with 20 mM inosine.
Inhibition of insulin-stimulated translocation of Glut 1 (Fig.
10A) and Glut 4 (Fig.
10B) to the PM by glucosamine was almost completely
prevented with 20 mM inosine. Glucosamine-induced
inhibition of Glut 1 (Fig. 10C) and Glut 4 (Fig.
10D) translocation from the LDM with insulin was also
largely prevented with inosine, as was the accumulation of Glut 4 in
the LDM under basal conditions. Restoration of insulin-stimulated tyrosine autophosphorylation of the insulin receptor at the PM and
tyrosine phosphorylation of IRS-1 in the LDM by inosine are shown in
Fig. 11, A and B,
respectively. Insulin activation of both autophosphorylation and
substrate tyrosine phosphorylation was almost completely restored with
inosine. ATP concentrations for the translocation and signaling
experiments with inosine are shown in Fig. 11C. The 60%
reduction in ATP by 2 mM glucosamine was largely but not
completely eliminated with 20 mM inosine (80% of control),
and this correlated well with the ability of inosine to increase but
not fully restore transporter translocation and insulin signaling.

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Fig. 10.
Prevention of glucosamine-induced inhibition
of Glut 1 (G1) and Glut 4 (G4) translocation by inosine. 3T3-L1
adipocytes were incubated for 1 h in the presence of 6 nM insulin in glucose-free DMEM containing nothing
(C), 2 mM glucosamine (GLN), or 2 mM glucosamine supplemented with 20 mM inosine
(GLN + INOSINE). Following three washes with PBS, cells were
incubated in the same media without insulin for 1.5 h. Cells were
then treated (hatched bars) or not treated
(black bars) with 1 µM insulin for
20 min prior to subcellular fractionation (see "Experimental
Procedures"). Data represent the mean ± S.E. of three
independent experiments. #, relative abundance differed significantly
(p < 0.05) in the stimulated state compared with the
basal state. *, relative abundance in the basal glucosamine samples
differed significantly compared with the basal control samples.
A, amount of Glut 1 transporter at the PM plotted relative
to insulin-stimulated control cells. B, amount of Glut 4 transporter at the PM plotted relative to insulin-stimulated control
cells. C, amount of Glut 1 transporter in the low density
microsomes plotted relative to basal control cells. D,
amount of Glut 4 transporter in the low density microsomes plotted
relative to basal control cells.
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Fig. 11.
Prevention of glucosamine-induced inhibition
of insulin signaling by inosine. 3T3-L1 adipocytes were incubated
for 1 h in the presence of 6 nM insulin in
glucose-free DMEM containing nothing (C), 2 mM
glucosamine (GLN), or 2 mM glucosamine
supplemented with 20 mM inosine (GLN + INOSINE).
Following three washes with PBS, cells were incubated in the same media
without insulin for 1.5 h. Cells were then treated
(hatched bars) or not (black
bars) with 1 µM insulin for 20 min prior to
subcellular fractionation (see "Experimental Procedures"). Data
represent the mean ± S.E. of three independent experiments. #,
relative abundance differed significantly (p < 0.05)
in the stimulated state compared with the basal state. *, relative
abundance in the insulin-stimulated glucosamine samples differed
significantly compared with the stimulated control samples.
A, amount of tyrosine-phosphorylated insulin receptor at the
PM plotted relative to insulin-stimulated control cells. B,
the amount of tyrosine-phosphorylated IRS-1 in the LDM plotted relative
to insulin-stimulated control cells. C, ATP concentrations
were determined as described (see "Experimental Procedures") and
plotted relative to control values. *, relative ATP concentrations in
the glucosamine cells differed significantly from that in control
cells.
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Effect of Overnight Glucosamine Treatment Carried Out in the
Presence of 5 mM D-Glucose on Cellular ATP
Levels--
In several published glucosamine studies (9, 11), 5 mM D-glucose was added to the media to ensure
cell viability, especially for longer glucosamine incubation times.
Therefore we decided to examine the effects of glucosamine treatment
under these conditions on intracellular ATP concentrations. In the
first experiment, cells were treated with DMEM containing varying
concentrations of glucosamine, 5 mM D-glucose,
and 6 nM insulin for 1 h. The cells were subsequently
washed three times with PBS and incubated for another 16 h in the
same medium but without insulin, and then cellular ATP levels were
determined. The results shown in Fig. 12A indicated that 5 mM D-glucose protected the cells completely from ATP depletion only at very low glucosamine concentrations (0.5-1
mM glucosamine). At 2 mM glucosamine and
higher, ATP levels decreased in a glucosamine
concentration-dependent manner with an ED50 of
6.08 mM. This ED50 was considerably higher than
the value we observed in the absence of D-glucose
(ED50 = 1.3 mM), and the absolute cellular ATP
levels at all glucosamine concentrations tested were also higher when
the media contained 5 mM D-glucose (Fig.
12A) compared with the levels we observed originally in the absence of D-glucose (see Fig. 6A for
comparison). In the second experiment, cells were treated with DMEM
containing varying concentrations of glucosamine, 5 mM
D-glucose, and 1 µM insulin for 16 h
prior to the ATP measurements. Insulin supplementation is often used in
many glucosamine experiments, since it has been reported that insulin
is a necessary cofactor for glucosamine-induced insulin resistance
(8-10). Interestingly, 5 mM D-glucose failed
to protect intracellular ATP levels even at 0.5 mM
glucosamine (Fig. 12B). Intracellular ATP decreased in the
presence of 0-2 mM glucosamine in an almost linear fashion
and then reached a lower limit of 0.4 relative to control cells (0 mM glucosamine). The same lower limit was observed in the
first experiment shown in Fig. 12A. These results indicate
that overnight insulin treatment shifted the glucosamine dose-response
curve to the left from an ED50 of 6.08 mM to an
ED50 of 0.95 mM, thus making 3T3-L1 adipocytes
more sensitive to glucosamine with regard to ATP depletion. As a
control, cells were treated for 16 h with DMEM containing 25 mM D-glucose and 1 µM insulin,
and we observed no change in ATP levels when compared with cells
treated for 16 h with 5 mM D-glucose, 0 mM glucosamine, and 1 µM insulin (data not
shown).

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Fig. 12.
Effect of overnight glucosamine treatment
supplemented with 5 mM D-glucose on cellular
ATP levels. A, 3T3-L1 adipocytes were incubated for
1 h in DMEM containing varying concentrations of glucosamine, 5 mM D-glucose, and 6 nM insulin. The
cells were subsequently washed three times with PBS and incubated for
another 16 h in the same media but without insulin, and then
ATP concentrations were determined as described under "Experimental
Procedures." B, cells were treated with DMEM containing
varying concentrations of glucosamine, 5 mM
D-glucose, and 1 µM insulin for 16 h
prior to the ATP measurements.
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DISCUSSION |
High glucose and insulin are known to cause insulin resistance in
the two target tissues of insulin, skeletal muscle and fat, that are
primarily responsible for glucose disposal (2, 4). The underlying
mechanism responsible for the defect is not well understood, but recent
studies have suggested that the hexosamine pathway may play an
important role (6). Glucosamine, which enters the hexosamine pathway
downstream from the rate-limiting step, has been routinely used to
study insulin resistance, and it is much more effective in eliciting
insulin desensitization than high glucose. In this study, the effects
of glucosamine on insulin-stimulated glucose transport were
investigated in 3T3-L1 adipocytes, one of the best characterized cell
lines used to study insulin action.
-insulin of 2-deoxyglucose
uptake was inhibited by glucosamine in a dose-dependent
manner with an ED50 that was similar to that reported by
Marshall et al. (5) for primary rat adipocytes. Our
subcellular fractionation results indicate that glucosamine inhibited
insulin-stimulated glucose uptake in 3T3-L1 adipocytes by blocking Glut
4 and Glut 1 translocation from the LDM to the PM. Effects on Glut 4 translocation by glucosamine have been previously observed in
vitro in primary rat adipocytes (9) and in vivo in
skeletal muscle (14). Our analysis of the signaling cascade showed that
glucosamine impaired insulin receptor autophosphorylation, IRS-1
phosphorylation, PI 3-kinase activity, and AKT activation by insulin.
In vitro phosphorylation experiments using semipurified
insulin receptor isolated from glucosamine-treated cells revealed that
the receptor itself was not directly affected. In one set of insulin
receptor preparations, we included tyrosine and serine phosphatase
inhibitors to preserve the in vivo phosphorylation state of
the receptor. In vitro phosphorylation results were the same
for receptors purified from control or glucosamine-treated cells but
activated in vitro with insulin (data not shown). Therefore, reductions in autophosphorylation and IRS-1 phosphorylation observed in vivo could not be due to a glucosamine-induced increase
in serine phosphorylation of the insulin receptor itself. We cannot rule out, however, that glucosamine may modulate receptor activity indirectly through another protein that is lost in the receptor isolation. These results are in apparent disagreement with the only
published report on the effects of glucosamine per se on early insulin signaling. Robinson et al. (11) found no
effect of glucosamine on in vitro substrate phosphorylation
of the synthetic substrate polyglutyr using semipurified insulin
receptor isolated from muscle stimulated in vivo with acute
insulin. However, insulin-stimulated tyrosine phosphorylation of the
insulin receptor, IRS-1 tyrosine phosphorylation, or any downstream
signaling step in the translocation cascade were not directly
investigated, nor were intracellular ATP concentrations reported. One
simple explanation for the discrepancy is that glucosamine affects
muscle and fat differently. Another possibility is that glucosamine
does affect early steps in insulin signaling in muscle such as IRS
phosphorylation, PI 3-kinase activity, or AKT activation and that
phosphorylation of the synthetic peptide substrate polyglutyr is not an
appropriate assay. Insulin receptors phosphorylate IRS-1 on multiple
tyrosine residues (40). Failure to phosphorylate a few residues may not
result in a significant reduction in the overall tyrosine
phosphorylation but may have a dramatic effect on the signaling
cascade.
Based upon the high correlation between decreases in ATP levels and
inhibition of insulin-stimulated 2-DOG uptake, the azide experiments,
and the prevention of glucosamine-induced insulin resistance by
inosine, our data strongly indicate that any desensitization of the
insulin-stimulated glucose transport effector system in 3T3-L1
adipocytes by glucosamine treatment is due entirely to effects on ATP
and not to increases in intracellular UDP-N-acetylhexosamine levels. What was most surprising was how tight the correlation was
between declines in ATP by glucosamine and induction of insulin resistance. Even what may be considered minor reductions in ATP (10-20%) resulted in insulin desensitization. Incubation of cells with high glucose and insulin overnight, however, had no effect on ATP
concentrations. We suspect that the decrease in ATP by glucosamine is a
result of rapid phosphorylation by hexokinase of newly transported
glucosamine. ATP is used but not quickly replenished, since the cells
are incubated in glucose-free media. This phenomenon is quite similar
to that of a recent study reported by Wang and Iynedjian (41) in which
they overexpressed glucokinase 20-fold in insulinoma cells. The
addition of D-glucose above 6 mM resulted in a
large accumulation of glucose 6-phosphate followed by a rapid decrease
in cellular ATP and eventual cell death. To the best of our knowledge,
our report is the first glucosamine study to report intracellular ATP
levels. It should also be considered that a 20% decline in ATP levels
measured from whole cell extracts may represent a much more significant
drop locally where the various phosphorylation reactions occur. Since
almost every step in the signaling cascade involves ATP utilization,
small effects early on will be amplified down the cascade. This was
apparent in our results, where IRS-1 phosphorylation was reduced 50%
but later events such as AKT activation and Glut 4 translocation were
completely inhibited. Even treating 3T3-L1 adipocytes overnight with
0.5 mM glucosamine, 5 mM D-glucose,
and insulin resulted in significant ATP depletions. Almost every
previously published glucosamine study could be reinterpreted in terms
of ATP depletion unless it was specifically ruled out. For example, a
recent study by Chen et al. (9) found that incubation of
primary fat cells with 2.5 mM glucosamine and 5 nM insulin for 4 h decreased 2-DOG transport by 50%
but had no effect on Glut 4 translocation. At longer glucosamine
incubation times (16 h), Glut 4 translocation was significantly
reduced. The authors speculate that glucosamine may affect the
intrinsic activity of the transporter at shorter incubation times. An
alternative explanation is that glucosamine causes a reduction in
cellular ATP, which could be initially localized near the site of
phosphorylation by hexokinase and then propagated with time throughout
the entire cell. Therefore, at short incubation times with glucosamine,
local reductions in ATP could cause the hexokinase reaction to become
rate-limiting for transport and reduce 2-DOG uptake but have no effect
on Glut 4 translocation. Glut 4 translocation could be inhibited at
longer glucosamine incubation times when ATP levels were decreased in
other regions of the cell. Insulin resistance has also been induced by
in vivo infusion of glucosamine into rats. Glucosamine
infusions were carried out for 7 h with 7 mM
D-glucose, 1.2 mM glucosamine, and 2.5 nM insulin (12, 13). Uridine administration also induced insulin resistance in these animals, and its effects were additive with
glucosamine. The authors concluded that reductions in insulin action by
glucosamine and uridine were mediated by the production of
UDP-N-acetylhexosamines. Since ATP levels were not reported, an alternative explanation is that both glucosamine and uridine could
decrease intracellular ATP levels and cause the insulin resistance. We
observed significant declines in ATP when 3T3-L1 adipocytes were
treated under similar glucosamine conditions (1 mM
glucosamine, 5 mM D-glucose, and insulin), and
although we never tested uridine directly, three high energy phosphates
are needed to generate a UDP-sugar from uridine. A recent study by Miles et al. (42) reported that troglitazone treatment can
reverse hyperglycemia-induced insulin resistance but not
glucosamine-induced insulin resistance in rats. These authors raised
the possibility that glucose may cause insulin resistance by a
mechanism independent of its entry into the hexosamine pathway. The
strongest evidence offered by Marshall et al. (5) that
increased flux through the hexosamine pathway is responsible for
glucose-induced insulin resistance was the observation that glucosamine
is 40 times more potent at inhibiting insulin-stimulated glucose
transport in fat cells than glucose. Our data suggest that glucosamine
may have acted by a different mechanism than high glucose altogether,
i.e. ATP depletion, and thus call into question the role of
increased flux through GFAT in the induction of insulin resistance.
Overexpression of GFAT, the enzyme catalyzing the rate-limiting step in
hexosamine biosynthesis, may be a more relevant approach to investigate
hexosamine involvement in insulin resistance if the effects induced by
glucosamine are found to be routinely correlated to ATP depletion.
Interestingly, however, when Chen et al. (9) overexpressed
GFAT in primary fat cells, they found it had little effect and
therefore they used glucosamine instead. On the other hand, GFAT
overexpression in fibroblasts by stable transfection does decrease
insulin sensitivity of glycogen synthase (15-17). In addition, the
transgenic study of Hebert et al. (18) showed that
overexpression of GFAT in muscle and fat did reduce glucose disposal.
Similarly, transgenic mice that overexpress Glut 1 in muscle are
insulin-resistant and have normal ATP levels but have increased GFAT
activity and UDP hexosamine levels (19-21).
Our results may have some profound mechanistic implications for the
pathogenesis of insulin resistance in human disease states such as type
2 diabetes mellitus. If our results with 3T3-L1 adipocytes are
representative of insulin-sensitive tissues in vivo, such as
liver, fat, and muscle, then relatively small reductions in intracellular ATP may translate into significant decreases in insulin
responsiveness. The implication is that any cellular defect that
results in decreased steady-state levels of ATP may induce a state of
insulin resistance.