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VA San Diego Healthcare System and Department of Medicine (0673) (T.P.C., L.C., S.N., S.M., R.R.H.), University of California, San Diego, La Jolla, California 92093; and Veterans Affairs Medical Center and Department of Medicine (D.A.M.), University of Mississippi Medical Center, Jackson, Mississippi 39216
Address all correspondence and requests for reprints to: Robert R. Henry, M.D., VA San Diego Healthcare System (V111G), 3350 La Jolla Village Drive, San Diego, California 92161. E-mail: rrhenry{at}vapop.ucsd.edu
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Introduction |
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Numerous other lines of evidence indicate that flux through the hexosamine pathway can influence glucose metabolism and insulin action (reviewed in Ref. 6). Overexpression of GFA, resulting in accumulation of glucosamine and other intermediates in the pathway, caused impairments in insulin stimulation of glycogen synthase (7). Direct addition of glucosamine, which enters cells via glucose transporters (5) and circumvents the rate limiting action of GFA, leads to insulin resistance in both in vivo (8, 9, 10) and in vitro (11, 12, 13) systems. In the hyperglycemic streptozotocin-diabetic rat (14) and the ob/ob mouse (15) either flux through the hexosamine pathway or GFA activity were found to be elevated in conjunction with insulin resistance. GFA activity was also found to be elevated in skeletal muscle biopsies from type 2 diabetic subjects (16).
We have studied human skeletal muscle cells maintained in culture under conditions that retain the characteristics of impaired insulin action and glucose metabolism displayed by diabetic subjects in vivo (17, 18). When cultured under euglycemic (5 mM) and normoinsulinemic (20 pM) conditions, there was no difference in GFA activity between cells from nondiabetic and type 2 diabetic subjects (19). However, GFA activity was upregulated by either hyperglycemia or hyperinsulinemia and more effectively by the combination (19), suggesting that the differences found in muscle biopsies may reflect the influence of the in vivo metabolic enviroment. In the current studies we directly tested the effects of altered flux through the hexosamine pathway, distal to GFA, on glucose transport, phosphorylation and glycogen synthesis, comparing the behavior of cultured muscle cells from nondiabetic and type 2 diabetic subjects.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human subjects
Both nondiabetic and type 2 diabetic subjects were recruited for
muscle biopsy. Glucose tolerance was determined from a 75 g oral
glucose tolerance test (20). Subject characteristics are summarized in
Table 1
. While both groups were, on
average, obese (BMI > 27), the diabetic group was significantly
more so. The response to the OGTT confirmed the insulin resistance of
the diabetic group. The experimental protocol was approved by the
Committee on Human Investigation of the University of California, San
Diego. Informed written consent was obtained from all subjects after
explanation of the protocol. Biopsy of the vastus lateralis muscle was
performed according to published procedures (17).
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-MEM containing 2% FBS, 1% fungibact, 100
U/ml penicillin, and 100 µg/ml streptomycin. When indicated, varying
concentrations of glucosamine were added during the last 2 days of
fusion. Fusion media was changed every 2 days to reduce glucose
depletion. All studies were carried out on first passage cultures. The
extent of differentiation was established by fluorescent microscopic
observation of multinucleation and striation patterns of skeletal
myosin fast (heavy chain), induction of sarcomeric actin protein,
induction of creatine kinase enzyme activity and CPKm mRNA abundance as
described in previous reports (17).
Glucose uptake assay
The procedure for glucose uptake measurements was modified from
that described by Klip et al. for L6 myocytes (21) and has
been described previously (22). Cells were washed free of treatments
added during culture and fresh serum-free media was added to the cells
together with insulin (0–33 nM) and the cells incubated
for 60–90 min in a 5% CO2 incubator before washing and
transport assay. An aliquot of the suspension was removed for protein
analysis using the Bradford method (23). The uptake of
L-glucose was used to correct each sample for the
contribution of diffusion. The accumulation of deoxyglucose within
cells measured in this assay is a result of both transport and
phosphorylation of the substrate. Throughout this report the term
glucose uptake will be used to refer to the combined measurement, not
the individual components.
Deoxyglucose phosphorylation
Free (nonphosphorylated) deoxyglucose was separated from
phosphorylated sugar by ion exchange chromatography (24). Incubation
with insulin and 3H-2-deoxyglucose was the same as for
glucose uptake but radioactivity was increased (to 0.3 µCi/reaction)
and only unlabeled L-glucose was present. Cells were washed
with PBS, 10% TCA added at 4 C, and cells scraped into microfuge
tubes. Precipitated material was collected by centrifugation and an
aliquot of the supernatant extracted 2x with diethyl ether. An aliquot
of the aqueous phase was loaded onto pasteur pipettes packed with ion
exchange resin (AG 1-X, 200–400 mesh, chloride form). Free
deoxyglucose was eluted with H2O. Deoxyglucose-6-phosphate
was eluted with 1 M HCl. The fractions were collected
directly into scintillation vials. Results are presented as percentage
(%) of total cell associated radioactivity in the free
(nonphosphorylated) fraction. Within the time frame (10 min) of the
deoxyglucose uptake assay there was no detectable 3H-DOG
incorporation into glycogen. For this reason it is most likely that
"free"DOG is unphosphorylated sugar. Glucosamine-6-phosphate
(GlcN-6-P) levels were measured in cell extracts as described
previously (19).
Membrane preparation
Cells for membrane preparation were grown in 100-mm dishes and
treated as described for activity assays. Total membranes were prepared
by the method developed by Walker et al. (25), as described
previously (22). The total membrane pellet was resuspended in
homogenization buffer and protein content determined.
Detection of glucose transporter proteins
Membrane preparations were diluted 1:1 in 2x Lamelli’s buffer
without ß-mercaptoethanol (26) and heated for 5 min at 90 C. Proteins
were separated on 10% SDS-PAGE gels and then transferred to
nitrocellulose (27). GLUT1 was identified using a rabbit polyclonal
antisera against the rat brain glucose transporter (RaGLUTRANS,
East Acres Biologicals). Polyclonal antisera specific for
GLUT4 (RaIRGT) and human GLUT3 were also employed. The second antibody
was antirabbit IgG conjugated with horseradish peroxidase. Immune
complexes were detected using an enhanced chemiluminescence kit.
Exposure was limited to the linear range of density as determined by
concentration curves established with human skeletal muscle total
membranes, included as an internal control. Quantitation was performed
with a scanning laser densitometer (ScanAnalysis,
Biosoft).
Glycogen synthase activity
The activity of glycogen synthase was measured as described in
detail previously (18) using modifications of the methods of Nuttall
et al. (28) and Thomas et al. (29).
Glycogen synthase activity was assayed at physiologic concentration of
substrate (0.3 mM UDP-[14C] glucose) in
parallel incubations with 0.1 and 10 mM
glucose-6-phosphate. For kinetic studies, total cell homogenates were
filtered through a Sephadex G-25 column, to remove small molecular
weight modifiers, and GS activity was measured at 0.1, 0.2, 0.6, 1.2,
and 5.0 mM UDPG in the absence or presence of 0.1,0.2, 0.3,
0.5, 2.0, and 10 mM glucose-6-phosphate (G-6-P) or
glucosamine-6-phosphate (GlcN-6-P). GS activity is expressed as nmol of
UDPG-glucose incorporated into glycogen·min-1·mg of
total protein, or as fractional velocity (FV), a percent (%) of the
ratio of activity at 0.1 mM G-6-P/10 mM
G-6-P.
Hexokinase activity
The same cell extracts were used for assay of glycogen synthase
and hexokinase (HK) activities. Spectrophotometric analysis of total
hexokinase activity was performed at 0.11 and 11 mM glucose
with the method described by Vestergaard et al. (30).
Potential effects of GlcN-6-P were tested by adding the metabolite
directly to the enzyme assay. A parallel sample was heated at 45 C for
60 min to destroy HKII activity and assayed for heat-stable activity.
HKII activity was calculated as the difference between total and
heat-stable activity. Activity is presented as nanomoles of glucose
converted/min/mg protein.
Glycogen synthesis
Glycogen synthesis was determined in differentiated myotubes
as14C-D- glucose incorporation into glycogen
during a 1 h incubation at 37 C (31). After incubation, cells were
rinsed 4 times with 4 C PBS and solubilized with 1 N NaOH
at 55 C for 1 h. An aliquot (100 µl) of the lysate was removed
for protein analysis. Lysates were neutralized with 10 N
HCL, boiled for 30 min and then cooled on ice. Glycogen was
precipitated with 95% ethanol and the pellet washed by resuspension
and precipitation. The final glycogen pellets were resuspended in 0.5
ml H2O, mixed with scintillation fluid and radioactivity
determined by liquid scintillation counting. Results are expressed as
nmol glucose converted to glycogen/mg protein·h.
Glycogen synthase immunobloting
Western blot analysis was performed by the method of Burnette
(32) as detailed previously (18). Glycogen synthase was identified
using an affinity purified polyclonal antibody raised in rabbits
against an oligopeptide (12-mer) specific for the carboxy terminal
sequence of glycogen synthase (a gift from Dr. L. Groop, Malmo,
Sweden). The secondary antibody was antirabbit IgG conjugated with
horseradish peroxidase. Proteins were visualized with the enhanced
chemiluminescence Western Blot Detection Kit (Amersham Pharmacia Biotech) and exposed to autoradiograph film (XAR-5,
Eastman Kodak Co., Rochester, NY). The intensity of the
bands was quantified by scanning laser densitometry.
Statistical analysis
Statistical significance was evaluated using Student’s
t test and two-tailed P values calculated. Paired
analysis was performed for comparisons of acute and chronic insulin and
glucosamine exposures in the same sets of cells. Significance was
accepted at the P < 0.05 level.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Basal uptake was reduced to 78
± 4% of control values (P < 0.0005).
Insulin-stimulated uptake was reduced to a similar extent (65 ±
3% of control, P < 0.0001). Glucosamine treatment
also led to impaired insulin responsiveness as the maximal insulin
stimulated increase in uptake was only 44 ± 13% of that in
control cells (P < 0.025).
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Glucosamine effects on glucose uptake were concentration-dependent
(Fig. 2). Nondiabetic and diabetic cells
were equally sensitive, with half of the maximal down-regulation
occurring at 2 mM for both basal and insulin-stimulated
uptake. Higher glucosamine concentrations (20 mM) were
deleterious, leading to a loss of cells (not shown).
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. Acute
insulin exposure did not alter total hexokinase activity. There was no
significant difference in hexokinase activity between nondiabetic and
diabetic cells. Heat labile activity, representing hexokinase II,
accounted for 48 ± 5% of the total activity; this proportion was
similar in diabetic and nondiabetic cells. Hexokinase activity was not
significantly influenced by glucosamine treatment in either nondiabetic
or type 2 diabetic cells (Table 2).
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). Because results
are calculated as a percentage of the total intracellular DOG, this
takes into account the reduced uptake of DOG (20–25% lower after
glucosamine treatment in these cells). Thus glucosamine treatment led
to absolute and relative decreases in deoxyglucose phosphorylation.
Glucosamine treatment also impaired phosphorylation in
insulin-stimulated cells. In cells from diabetic subjects glucosamine
treatment also increased the fraction of free DOG in both the basal and
insulin-stimulated conditions (Table 2). Thus glucosamine was
similarily capable of inhibiting glucose phosphorylation in both
nondiabetic and diabetic muscle cells, even as isolated hexokinase
activity was unaltered. One possible cause for this discrepancy could
be the influence of intracellular metabolites, as suggested above,
which would be diluted upon cell extraction. In fact, addition of
GlcN-6-P (2 mM) directly to the assay inhibited total
hexokinase activity by 26 ± 10% (P < 0.01). To
test this question further, GlcN-6-P levels in cells were determined
and found to decrease with glucosamine treatment (0.16 ± 0.02
vs. 0.12 ± 0.03 nmol/mg protein, control
vs. treated, respectivly, P < 0.025),
suggesting that added glucosamine is primarily converted to UDP-GlcNAc
(9) and that direct inhibition of hexokinase by intracellular GlcN-6-P
may contribute little to the effects shown in Table 2. These values are
for nondiabetic and diabetic cells combined, as there were no
consistent differences between groups.
Regulation of glucose transporter expression
One possible mechanism by which glucosamine treatment could lead
to down-regulation of glucose uptake would be to reduce glucose
transporter protein expression. Total membranes were prepared from
nondiabetic cells and GLUT1, GLUT3, and GLUT4 expression measured by
Western blotting. Glucose transport was measured in parallel in cells
from the same subjects; the treatment conditions caused approximately
30% reductions in both basal and insulin-stimulated transport (not
shown). Glucosamine treatment had no consistent effect on either GLUT1
(97 ± 15% of untreated control, n = 6), GLUT3 (102 ±
30%, n = 4) or GLUT4 (107 ± 12%, n = 4) protein
expression. It should be emphasized that these measurements were made
in total membranes and could not distinguish between cell surface and
intracellular pools of transporters.
A major role of the hexosamine pathway is to provide substrates for protein glycosylation (34). Both GLUT1 and GLUT4 are highly glycosylated proteins (35) and it is possible that increased flux through the pathway could lead to altered transporter glycosylation. If this occurred, it was not detectable as a change in transporter mobility with the methods used (not shown).
Influence of glucosamine on glycogen synthase activity
The activity of the rate limiting enzyme for nonoxidative glucose
metabolism in muscle, glycogen synthase, was measured after treatment
under conditions that caused maximal down-regulation of uptake. As
reported previously with other subjects (18), both basal glycogen
synthase activity at 0.1 mM G-6-P and fractional velocity
were lower (P < 0.05) in diabetic muscle cells
compared with nondiabetics (Table 3). In
contrast to the effect on transport/phosphorylation, glucosamine (10
mM) treatment resulted in an increase in the fractional
velocity (FV) of glycogen synthase (Fig. 3). The extent of stimulation was similar
in nondiabetic (129 ± 14% of control, P < 0.05)
and diabetic (135 ± 8%, P < 0.05) cells.
Insulin was still able to acutely stimulate glycogen synthase. In fact,
in nondiabetic cells the insulin effect (to 175 ± 17% of basal)
was augmented after glucosamine treatment, compared with control
(145 ± 8% stimulation, P < 0.05), suggesting
that the insulin and glucosamine effects were additive. Relative
insulin stimulation in diabetic cells was not different before
(164 ± 13%) or after (161 ± 14%) glucosamine treatment.
This effect on glycogen synthase activity was dose dependent, with half
maximal effects at 2 mM glucosamine in both nondiabetic and
diabetic cells (not shown), similar to that for down-regulation of
glucose uptake. The glucosamine-mediated increase in glycogen synthase
is most likely due to a change in the activation state of the enzyme as
neither total activity (measured at 10 mM G-6-P) nor
glycogen synthase protein (as determined by Western blot), were altered
by glucosamine treatment (not shown). These later results were observed
in both nondiabetic and type 2 cells.
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), it was only a fraction (59 ± 6%,
P < 0.05) of the activity measured with 0.1
mM G-6-P. Activity at 10 mM GlcN-6-P was
similarly reduced compared with 10 mM G-6-P (49 ± 7%
of 10 mM G-6-P value). The fractional velocity at 0.1
mM GlcN-6-P was half that at 0.1 mM G-6-P.
Thus, GlcN-6-P is only half as effective an allosteric activator of
glycogen synthase as G-6-P. Similar results were obtained in diabetic
cells (Table 3), where activity at both 0.1 mM GlcN-6-P
(52 ± 6% of G-6-P, P < 0.05) and 10
mM GlcN-6-P (48 ± 7%) were lower compared with
G-6-P.
The results in Table 3 suggest that elevations in GlcN-6-P, arising
from increased flux through the hexosamine pathway, could contribute to
stimulation of glycogen synthase. However, this mechanism need not
account for all of the stimulatory effect, for augmented synthase
activity in glucosamine-treated cells was observed even after
chromomatographic removal of low molecular weight modulators (Fig. 4). In nondiabetic muscle cells, there
was a tendency for both basal and insulin-stimulated synthase
activities in glucosamine-treated cells to remain higher than controls
after chromatography, though these differences did not attain
statistical significance (P = 0.078). In diabetic cells
the residual effects of glucosamine treatment on synthase were
significant (P < 0.05) in both the basal and
insulin-stimulated states (Fig. 4). Thus, nonallosteric mechanisms may
also be active. These could involve covalent modification of the
synthase. The fact that intracellular GlcN-6-P levels actually fell
with glucosamine treatment indicate that nonallosteric mechanisms may
represent the major means of synthase regulation in this situation.
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). A similar decrease in cellular glycogen following
treatment was observed in diabetic cells (from 0.94 ± 0.09 to
0.67 ± 0.09, P < 0.002).
Net glucose incorporation into glycogen represents the balance
between glucose uptake, glycogen synthesis and breakdown during the
time of the assay. This activity was measured in control and
glucosamine-treated human muscle cells (Fig. 5), under conditions where uptake was
reduced by approximately 30%, and synthase activity was elevated by
approximately 30%. In nondiabetic cells, basal glucose incorporation
was reduced by glucosamine treatment (68 ± 8% of control,
P < 0.025), as was insulin-stimulated activity
(55 ± 4% of control, P < 0.0001). While insulin
stimulation of glycogen synthesis was retained in glucosamine-treated
cells (136 ± 12% of basal, P < 0.02), both the
absolute and relative increases were less than those seen in control
cells (165 ± 13% of basal). Similar behavior was observed in
type 2 diabetic cells: both basal (74 ± 9% of control,
P < 0.05) and insulin-stimulated (72 ± 6%,
P < 0.02) synthesis were reduced following glucosamine
treatment. Relative insulin stimulation was comparable in control
(148 ± 18% of basal) and glucosamine-treated (145 ± 14%)
diabetic cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Studies in humans revealed that GFA activity in skeletal muscle biopsies was elevated in type 2 diabetic subjects (16). Such an elevation in GFA activity has the potential to exacerbate the deleterious effects of hyperglycemia by directing more glucose into the hexosamine pathway. Such differences were not seen when cultured skeletal muscle cells from nondiabetic and type 2 diabetic subjects were compared (19). Because GFA activity in cultured muscle cells can be regulated by media glucose and insulin levels (19), it is likely that the differences seen in biopsies reflect the influence of the in vivo metabolic environment. Interestingly, the relationship between insulin-stimulated whole body glucose disposal and GFA activity in cultured muscle cells from the same individuals was different in nondiabetic and type 2 diabetic subjects (19); suggesting that the link between production of GlcN-6-P and the response of glucose uptake to flux through the hexosamine pathway may be altered in diabetes. Further evidence for differences in the sensitivity of nondiabetic and diabetic skeletal muscle to glucose were observed in the effects of chronic hyperglycemia on glucose uptake in cultured muscle cells: hyperglycemia was able to down regulate uptake in diabetic cells while having no effect in nondiabetic cells (22). The current studies were intended to see if such differences extended to processes beyond GFA.
The ability of glucosamine pretreatment to down-regulate glucose uptake
in cultured human muscle cells (Fig. 1) is similar to the results
observed in primary cultured rat adipocytes (5, 43), fibroblasts (13),
intact rats (41), and isolated muscles (11, 13). A report in cultured
L6 muscle cells represents the one instance where disruption of the
hexosamine pathway (inhibition of GFA) failed to perturb glucose
transport (12). The glucosamine-induced reduction of glucose transport
in cultured human muscle cells occurred in the absence of any change in
total expression of GLUT1, GLUT3, or GLUT4 proteins. A similar lack of
effect of glucosamine on transporter levels was seen after in
vitro treatment of rat adipocytes (43) or muscle (11, 13), or in
skeletal muscle following glucosamine infusion (41). Such observations
led to the hypothesis that either insulin signaling or transporter
translocation was the site of glucosamine-induced insulin resistance
and glucose toxicity. Impaired GLUT4 translocation following
glucosamine treatment has been demonstrated in skeletal muscle (41) and
3T3-L1 adipocytes (44). The relative diminution of insulin
responsiveness of transport is evidence that increased flux through the
hexosamine pathway also leads to a measure of insulin resistance.
Glucosamine infusion has recently been shown to impair selected early
events in insulin signaling (45, 46), but not others (13, 46); this
topic certainly merits further study in human muscle. In addition, the
fact that both basal and insulin-stimulated transport are impaired
suggests that some basic aspect of transporter function is also
influenced by glucosamine. Possibilities include transporter activity
(44) and orientation in the membrane.
Both GLUT1 and GLUT4 are extensively glycosylated on a single asparagine residue (47). As a major product of the hexosamine pathway is UDP-N-acetylglucosamine, an important substrate for protein glycosylation (34), it is possible that elevated flux through the hexosamine pathway, with resultant increases in UDP-N-acetylglucosamine, could lead to altered transporter glycosylation. Because transporter glycosylation influences the affinity, activity, stability, and subcellular distribution of glucose transporters (47, 48, 49), alterations in transporter glycosylation could contribute to the impairments in transport activity resulting from glucosamine treatment, especially the proposed defect in insulin-stimulated translocation. Changes in transporter glycosylation are often detected by changes in electrophoretic mobility (35). No such differences were observed for either GLUT1 or GLUT4 after glucosamine treatment (not shown). Baron et al. also reported no change in rat skeletal muscle GLUT4 mobility after glucosamine infusion (41). These results suggest that transporter glycosylation is not grossly altered by glucosamine. However, more sensitive and specific analysis is necessary to rule out subtle changes in the extent or identity of transporter glycosylation. In addition, incorporation of labeled glucosamine into GLUT4-containing vesicles from skeletal muscle is elevated following glucosamine infusion (50), suggesting that glycosylation of proteins other than GLUT4 may be influenced and contribute to changes in glucose transport.
Beyond the reduction in glucose uptake, glucosamine treatment was also
found to reduce glucose phosphorylation (Table 2). This is in agreement
with the report of Furnsinn et al. (11), who found that
glucosamine treatment of isolated rat soleus muscles decreased both
glucose transport and phosphorylation. It has been demonstrated that
hexokinase II activity and expression are reduced in skeletal muscle
from type 2 diabetic subjects (51, 52), supporting the possibility that
both glucose phosphorylation and transport are impaired in diabetes,
just as they are by glucosamine. Evidence suggests that these defects
in hexokinase II are reflective of the in vivo metabolic
environment of the subject (52). Thus, it is not surprising that we
found no difference in either total or hexokinase II activity between
cells from nondiabetic and diabetic subjects (Table 2) because cells
from all subjects were cultured under matched euglycemic and
normoinsulinemic conditions. Hexokinase activity in cells did not
respond to acute insulin treatment. In vivo responses of
hexokinase II activity to insulin infusion are modest (51, 53), require
greater than 3 h of insulin exposure (51), and follow increases in
hexokinase II expression (54). It is likely that the time for insulin
treatment of cultured muscle cells (1 h) was too brief to observe
similar changes, even though this time was sufficient for full
activation of glycogen synthase. Recent evidence has indicated that
more rapid insulin effects on hexokinase II in skeletal muscle involve
a redistribution from the cytosolic to the particulate fraction (55);
such a change would not be detectable in our total cell extracts.
It was initially unexpected that the same conditions of glucosamine
treatment that resulted in significant impairments in deoxyglucose
phosphorylation had no effect on hexokinase activity measured in cell
extracts (Table 2). As mentioned previously, measurement of
deoxyglucose phosphorylation reflects the combined roles of substrate
delivery (transport) and hexokinase activity, as well as the influence
of intracellular modulators. One of the most important might be
GlcN-6-P because it can cause feedback inhibition of hexokinase (56).
In intact cells GlcN-6-P might attain levels sufficient to inhibit
hexokinase, yet be diluted to ineffective levels during extraction
before the hexokinase assay, an idea supported by the observation that
GlcN-6-P can directly inhibit hexokinase from cultured muscle cells. In
addition, GlcN-6-P levels attained in rat muscle following glucosamine
infusion were capable of decreasing hexokinase activity (57). These
results, together with the fact that hexokinase itself is unaltered
following glucosamine treatment, suggests that impaired glucose
phosphorylation is due to the intracellular environment, including
allosteric factors, and not to changes in hexokinase expression or
covalent modification of the enzyme that survive cell lysis.
Unlike the reductions in glucose transport/phosphorylation following
glucosamine treatment, glycogen synthase fractional velocity was
augmented following treatment (Fig. 3). Similar increases in glycogen
synthase activity were also observed in glucosamine treated HIRc cells
(13) and rat-1 fibroblasts overexpressing GFA (7). In all cases, total
glycogen synthase activity was unaltered; rather it was the activation
state of the enzyme that was effected (7). While GlcN-6-P can serve as
an allosteric activator of glycogen synthase (7 and Table 3), both the
results in human muscle cultures (Fig. 4) and GFA-over expressing
fibroblasts suggest (7) that additional mechanisms are also responsible
for stimulation of glycogen synthase activity. As glycogen synthase
activity is regulated by phosphorylation/dephosphorylation (58), it is
possible that flux through the hexosamine pathway may influence a
kinase or phosphatase acting on glycogen synthase.
Skeletal muscle from type 2 diabetic subjects displays impairments in glucose transport/phosphorylation (59) and glycogen synthase activity (60). These defects are most often preserved in cultured muscle cells from diabetic subjects (17, 18). The effects of glucosamine treatment on glucose transport, reduction in activity, and insulin responsiveness with no change in GLUT4 expression are similar to those of diabetes, so it is quite possible that flux through the hexosamine pathway could contribute to this aspect of diabetes in muscle. However, the relative effects of diabetes and glucosamine on glycogen synthase are opposite, suggesting that increased flux through the hexosamine pathway alone does not create a complete model for the diabetic state. It may also be that the glucosamine effect on transport/phosphorylation is the predominant influence as both total glycogen stores and net glycogen synthesis are reduced: in light of reduced uptake and augmented synthase, it appears to be uptake that determines nonoxidative glucose utilization. The activation of synthase observed in response to glucosamine could be an attempt to compensate for the other negative effects of hyperglycemia, including those on transport.
One interesting aspect of the effect of glucosamine on glucose uptake
is the similar sensitivity of nondiabetic and diabetic cells to the
sugar (Fig. 2), unlike the behavior in response to glucose (22). Such
results suggest that the increased sensitivity of diabetic muscle to
glucose resides before the generation of glucosamine. The most likely
candidate for this step would be GFA. In parallel behavior,
overexpression of GFA in rat fibroblasts increased sensitivity to the
ability of glucose to cause insulin resistance (61). There were no
major differences between nondiabetic and type 2 diabetic muscle cells
with regard to the various effects of glucosamine treatment
observed.
In summary, increased flux through the hexosamine pathway, caused in the current situation by glucosamine treatment, can impair glucose transport/phosphorylation and glycogen synthesis and lead to impaired insulin responsiveness, if not total insulin resistance. While increased flux through the hexosamine pathway might not generate all the features of the diabetic state in cultured muscle cells, it could certainly play a large role. The similar sensitivity of nondiabetic and diabetic muscle to glucosamine suggests that the greater negative impact of hyperglycemia in diabetes may be mediated before the formation of glucosamine, possibly at GFA.
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Footnotes |
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Received February 18, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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L. Ma, W. A. Rudert, J. Harnaha, M. Wright, J. Machen, R. Lakomy, S. Qian, L. Lu, P. D. Robbins, M. Trucco, et al. Immunosuppressive Effects of Glucosamine J. Biol. Chem., October 11, 2002; 277(42): 39343 - 39349. [Abstract] [Full Text] [PDF]
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 D. A. McClain, W. A. Lubas, R. C. Cooksey, M. Hazel, G. J. Parker, D. C. Love, and J. A. Hanover Altered glycan-dependent signaling induces insulin resistance and hyperleptinemia PNAS, August 6, 2002; 99(16): 10695 - 10699. [Abstract] [Full Text] [PDF]
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 M. Federici, R. Menghini, A. Mauriello, M. L. Hribal, F. Ferrelli, D. Lauro, P. Sbraccia, L. G. Spagnoli, G. Sesti, and R. Lauro Insulin-Dependent Activation of Endothelial Nitric Oxide Synthase Is Impaired by O-Linked Glycosylation Modification of Signaling Proteins in Human Coronary Endothelial Cells Circulation, July 23, 2002; 106(4): 466 - 472. [Abstract] [Full Text] [PDF]
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M. Gaster, I. Petersen, K. Hojlund, P. Poulsen, and H. Beck-Nielsen The Diabetic Phenotype Is Conserved in Myotubes Established From Diabetic Subjects: Evidence for Primary Defects in Glucose Transport and Glycogen Synthase Activity Diabetes, April 1, 2002; 51(4): 921 - 927. [Abstract] [Full Text] [PDF]
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L. M. Perriott, T. Kono, R. R. Whitesell, S. M. Knobel, D. W. Piston, D. K. Granner, A. C. Powers, and J. M. May Glucose uptake and metabolism by cultured human skeletal muscle cells: rate-limiting steps Am J Physiol Endocrinol Metab, July 1, 2001; 281(1): E72 - E80. [Abstract] [Full Text] [PDF]
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 A. R. Shikhman, K. Kuhn, N. Alaaeddine, and M. Lotz N-Acetylglucosamine Prevents IL-1{{beta}}-Mediated Activation of Human Chondrocytes J. Immunol., April 15, 2001; 166(8): 5155 - 5160. [Abstract] [Full Text] [PDF]
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M. Gaster, A. Handberg, H. Beck-Nielsen, and H. D. Schroder Glucose transporter expression in human skeletal muscle fibers Am J Physiol Endocrinol Metab, September 1, 2000; 279(3): E529 - E538. [Abstract] [Full Text] [PDF]
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L. Rossetti Perspective: Hexosamines and Nutrient Sensing Endocrinology, June 1, 2000; 141(6): 1922 - 1925. [Full Text] [PDF]
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