From the Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110
Received for publication, November 22, 2000, and in revised form, March 8, 2001
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ABSTRACT |
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Muscles and fat cells develop insulin resistance
when exposed to high concentrations of glucose and insulin. We used an
isolated muscle preparation incubated with high levels of glucose and
insulin to further evaluate how glucose-induced insulin resistance
(GIIR) is mediated. Incubation with 2 milliunits/ml insulin and 36 mM glucose for 5 h resulted in an ~50%
decrease in insulin-stimulated muscle glucose transport. The decrease
in insulin responsiveness of glucose transport induced by glucose was
not due to impaired insulin signaling, as insulin-stimulated
phosphatidylinositol 3-kinase activity and protein kinase B
phosphorylation were not reduced. It has been hypothesized that entry
of glucose into the hexosamine biosynthetic pathway with accumulation
of UDP-N-acetylhexosamines (UDP-HexNAcs) mediates GIIR.
However, inhibition of the rate-limiting enzyme GFAT
(glutamine:fructose-6-phosphate amidotransferase) did not protect
against GIIR despite a marked reduction of UDP-HexNAcs. The mRNA
synthesis inhibitor actinomycin D and the protein synthesis inhibitor
cycloheximide both completely protected against GIIR despite the
massive increases in UDP-HexNAcs and glycogen that resulted from
increased glucose entry. Activation of AMP-activated protein kinase
also protected against GIIR. These results provide evidence that GIIR
can occur in muscle without increased accumulation of hexosamine
pathway end products, that neither high glycogen concentration nor
impaired insulin signaling is responsible for GIIR, and that synthesis
of a protein with a short half-life mediates GIIR. They also suggest
that dephosphorylation of a transcription factor may be involved in the
induction of GIIR.
Hyperglycemia can lead to the development of insulin resistance, a
phenomenon thought to contribute to impaired insulin action in diabetes
(1-3). Glucose-induced insulin resistance, also referred to as
"glucose toxicity," has been studied in a number of experimental models. These include rats infused with high concentrations of glucose
(4-6), perfused rat hind limb muscles (7-10), isolated muscles
incubated in vitro (11, 12), primary cultures of rat adipocytes (13-15), and muscles of transgenic mice overexpressing GLUT1 (16, 17).
There are currently three disparate hypotheses regarding the mechanism
responsible for glucose-induced insulin resistance of glucose
transport. One is that accumulation of large amounts of glycogen, as a
result of glucose flooding into the cell, causes insulin resistance (7,
11, 18), possibly as a consequence of association of GLUT4-containing
vesicles with glycogen particles (19, 20). A second hypothesis is that
glucose toxicity is mediated by activation of protein kinase C (5, 21,
22) resulting in increased serine and threonine phosphorylation of the
insulin receptor, with a decrease in insulin stimulation of insulin
receptor tyrosine kinase (21, 23-25). The third is that glucose-induced insulin resistance is due to an increased flux of
glucose into the hexosamine biosynthetic pathway with accumulation of
UDP-N-acetylhexosamines (UDP-HexNAcs) (13, 14, 26-29). In addition to disagreement regarding the mechanism, there is controversy regarding whether rapid entry of glucose into the cell, mediated by
high concentrations of both glucose and insulin (30) or just exposure
to a high concentration of glucose (8, 10), is responsible for
glucose-induced insulin resistance.
In this study we used an in vitro skeletal muscle
preparation to evaluate these hypotheses and to further examine the
mechanism responsible for glucose-induced insulin resistance. Our
results do not support any of the current hypotheses regarding the
etiology of glucose toxicity. They show that the decrease in insulin
responsiveness of glucose transport results from a rapid influx of
glucose and suggest that increased expression of a protein with a short
half-life mediates the insulin resistance.
Materials--
The antiphospho-protein kinase B
(PKB)1 Ser473 and
Thr308 polyclonal antibodies were from Upstate
Biotechnology. Horseradish peroxidase-conjugated donkey anti-rabbit IgG
was obtained from Jackson ImmunoResearch Laboratories.
[14C]Mannitol,
3-O-[3H]methyl-D-glucose,
and [ Animals--
This research was approved by the Animal Studies
Committee of Washington University. Male Wistar rats weighing 100-140
g were obtained from Charles River and fed Purina chow and water until the evening before an experiment, when food was removed at ~6:00 p.m.
Induction of Insulin Resistance in Vivo--
Rats were
anesthetized with an intraperitoneal injection of sodium pentobarbital,
5 mg/100 g of body weight, and a polyethylene catheter was placed in an
external jugular vein. The catheter was tunneled under the skin,
exteriorized between the shoulder blades, sutured in place, and filled
with saline. The surgical incision was closed, and the externalized
catheter was covered with a jacket (Harvard Apparatus). After the rats
had recovered from surgery for 3 days, the catheters were threaded
through a flexible spring tether attached to a swiveling infusion
device (Harvard Apparatus) that allowed the animals to move freely
during the infusion. The rats were infused with either 50% glucose at an infusion rate of 50 mg glucose/kg of body weight/min or with 0.9%
saline for 24 h. At the end of the infusion period, the rats were
anesthetized with sodium pentobarbital, 5 mg/100 g of body weight.
Blood samples were obtained for measurement of glucose and insulin
(31). The epitrochlearis muscles were dissected out and incubated for
60 min with 60 microunits/ml or 2 milliunits/ml insulin prior to
measurement of glucose transport activity. After removal of the
epitrochlearis muscles, insulin was infused at a rate of 7.2 milliunits/min/100 g of body weight for 30 min to induce a maximal
insulin stimulus; glucose was also infused to prevent hypoglycemia.
After 30 min of insulin infusion, the gastrocnemius/plantaris muscle
group was excised for determination of GLUT4 in the sarcolemma.
Induction of Insulin Resistance in Vitro--
Epitrochlearis
muscles were placed in 2 ml of oxygenated Krebs-Henseleit bicarbonate
buffer (KHB) with or without 2 milliunits/ml insulin, 0.1%
radioimmunoassay grade bovine serum albumin, various concentrations of glucose, and sufficient mannitol so that the concentration of glucose plus mannitol was 40 mM.
Routinely, 36 mM glucose was used to induce muscle insulin
resistance, and 5 mM glucose was used as the control. The
other additions to the incubation medium are described for each
experiment under "Results" and/or the figure legends. The muscles
were incubated with shaking at 35 °C, and the flasks were gassed
continuously with 95% O2, 5% CO2. During 5-h
long incubations, the muscles were placed in fresh incubation medium
after 2.5 h. After the incubations some muscles were blotted and
used for measurement of metabolites, while others were used for
measurement of glucose transport activity.
Measurement of Glucose Transport Activity--
After the initial
incubation period, the muscles were quickly rinsed twice in KHB and
transferred to flasks containing 2 ml of KHB with 40 mM
mannitol, with the same concentration of insulin as in the initial
incubation, and incubated with shaking at 30 °C to remove glucose.
This procedure was repeated once. Glucose transport activity was then
measured as described previously (32, 33) using either
2-deoxy-D-glucose (2DG) (33) or
3-O-methyl-D-glucose (3MG) (32).
Plasma Membrane Preparation--
The increase in GLUT4 protein
in the plasma membrane in response to a maximally effective insulin
stimulus was compared in muscles of the rats infused for 24 h with
50% glucose or 0.9% saline. The muscles were processed and
homogenized as described previously (34), the homogenate was
centrifuged for 20 min at 48,000 × g, and the pellet
was used to prepare a plasma membrane fraction as described by
Hirshman et al. (35). GLUT4 immunoreactivity in the
whole homogenate and the plasma membrane fraction were determined by
Western blot analysis as previously described (36) using a rabbit
polyclonal antibody directed against the COOH terminus of GLUT4 (F349).
Measurement of Muscle Metabolites--
Muscle extracts were
prepared as described previously (18) and analyzed for UDP-GlcNAc and
UDP-GalNAc by high-performance liquid chromatography using the
method of Holstege et al. (37). Glycogen was measured on
perchloric acid homogenates of muscle using the amyloglucosidase method
(38). Muscle glucose 6-phosphate,2-deoxyglucose-6-phosphate and ATP
concentrations were measured fluorometrically (39) on neutralized
perchloric acid extracts (33).
Assessment of Insulin Signaling--
Muscle samples were
homogenized in 50 mM HEPES (pH 7.4), 150 mM
NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM
MgCl2, 1.0 mM aprotinin (10 µg/ml), leupeptin
(10 µg/ml), pepstatin (0.5 µg/ml), and phenylmethylsulfonyl
fluoride (2 mM). Homogenates were incubated with
end-over-end rotation at 4 °C for 60 min and then centrifuged at
200,000 × g for 50 min at 4 °C.
For analysis of PI 3-kinase activity associated with phosphorylated
tyrosine, aliquots of supernatant containing 1 mg of protein were
immunoprecipitated overnight with end-over-end rotation at 4 °C in
the presence of 40 µl of monoclonal antiphosphotyrosine antibody
coupled to protein A-Sepharose. Immunocomplexes were collected by
centrifugation and washed, suspended in assay medium, and analyzed for
PI 3-kinase activity as described by Goodyear et al.
(40).
For quantification of phosphorylated PKB, aliquots of the
200,000 × g supernatant were treated with 2× Laemmli
sample buffer containing 100 mM dithiothreitol and boiled
for 5 min. Samples (80 µg of protein) were subjected to
SDS-polyacrylamide gel electrophoresis (10% resolving gel) and were
then transferred to nitrocellulose membranes. The membranes were
blocked in 5% nonfat dry milk in Tris-buffered saline containing 0.1%
Tween 10 (TBST), pH 7.5, overnight. The membranes were rinsed in TBST
and incubated with either antiphospho-PKB Ser473
antibody or antiphospho-PKB Thr308 antibody for 4 h.
The membranes were rinsed in TBST and incubated with horseradish
peroxidase-conjugated donkey anti-rabbit IgG for 60 min. Antibody-bound
protein was visualized by ECL. The intensity of the bands corresponding
to phosphorylated PKB was assessed by densitometry.
Statistical Analyses--
Results are expressed as mean ± S.E. The significance of differences between two groups was assessed
using Student's unpaired t test. For multiple comparisons,
the significance was evaluated by analysis of variance. A
Newman-Keul's post hoc test was used to locate significant mean differences.
Effect of Glucose Infusion for 24 h--
In our initial
experiment, we gave rats glucose by intravenous infusion for 24 h.
The glucose infusion resulted in large increases in plasma glucose and
insulin, to levels in the range sometimes found in untreated patients
with type 2 diabetes (Table I). Muscle glycogen was also increased. Fig. 1 shows
insulin-stimulated 2DG transport rates in epitrochlearis muscles
studied in vitro after the 24-h infusion period. The
increase in 2DG transport above basal induced by either 60 microunits/ml or 2 milliunits/ml of insulin was ~60% smaller in
muscles from the glucose-infused as compared with the saline-infused
animals. (In our epitrochlearis muscle preparation, glucose
transport is stimulated maximally by an insulin concentration of ~500
microunits/ml.)
The GLUT4 protein content of the plasma membrane fraction was compared
in the muscles of 24-h glucose- and saline-infused rats. A maximally
effective insulin stimulus was provided by means of an intravenous
insulin infusion prior to harvesting of muscles (34). Hypoglycemia was
prevented by glucose infusion. Although the plasma membrane fraction
prepared by the subcellular fractionation procedure is heavily
contaminated with GLUT4 containing vesicles (41), it seems clear that
the insulin-induced increase in GLUT4 in the plasma membrane fraction
was ~50% smaller in the glucose-infused group (Fig.
2).
Induction of Muscle Insulin Resistance in Vitro--
To examine
the mechanisms by which rapid glucose entry into muscle cells causes a
decrease in insulin responsiveness, we developed an in vitro
model in which this phenomenon can be studied under controlled
conditions in a reasonable time period. Fig.
3 shows the effect of incubating rat
epitrochlearis muscles for 5 h with 2 milliunits/ml insulin and
various concentrations of glucose. Insulin responsiveness decreased
with increasing glucose concentration. Incubation of epitrochlearis
muscles with 36 mM glucose and insulin for 5 h
resulted in as great a decrease in insulin responsiveness as that
induced by the 24-h glucose infusion used in our initial experiment.
This experimental protocol, i.e. incubation of muscles for
5 h with 36 mM glucose and 2 milliunits of insulin,
was therefore routinely used to induce insulin resistance in subsequent
experiments.
Epitrochlearis muscles tolerated the 5 h of in vitro
incubation well. This was evidenced by maintenance of a normal ATP
concentration in muscles, rapid glycogen synthesis (see below), and
normal extracellular space. ATP concentration was the same in muscles
incubated for 5 h with 5 mM (4.4 ± 0.2 µmol/g)
or 36 mM glucose (4.6 ± 0.2 µmol/g) compared with
4.6 ± 0.3 µmol/g for muscles clamp-frozen in situ (values are means ± S.E. for 6 muscles/group). Insulin-stimulated glucose transport activity was also normal after 5 h, as evidenced by the finding that 3MG transport rate was not significantly different after 5 h, as compared with 1 h of incubation with 5 mM glucose plus 2 milliunits of insulin (Table
II).
Exposure of Muscles to High Glucose in the Absence of
Insulin--
It has been reported that exposure of muscles to a high
concentration of glucose in the absence of insulin causes insulin resistance (8). We therefore examined the effect of exposure of muscles
to 36 mM glucose in the absence of insulin. Muscles were
incubated for 5 h without insulin and then incubated with 2 milliunits/ml insulin for the 20-min period during which glucose was
washed out of the extracellular space and during the measurement of 3MG
transport. As shown in Fig. 4, muscles
incubated with 36 mM glucose for 5 h without insulin
showed no decrease in insulin responsiveness, whereas muscles that
underwent the same procedure in the presence of insulin became markedly
insulin-resistant. Thus, the insulin resistance is due to rapid entry
of large amounts of glucose into muscle rather than to exposure to
either a high glucose or a high insulin concentration. That a high
insulin concentration per se is not responsible for the
insulin resistance is evidenced by the finding that muscles exposed to
5 mM glucose and 2 milliunits/ml insulin for 5 h did
not become insulin-resistant.
Evaluation of the Role of Hexosamine Pathway End
Products--
Studies by Marshall and co-workers (13, 14) on fat cells
have led to the concept that glucose toxicity-induced insulin resistance is due to the accumulation of hexosamine pathway end products. One finding that led to this conclusion was that inhibition of glutamine:fructose-6-phosphate amidotransferase (GFAT), the rate-limiting enzyme in the hexosamine biosynthetic pathway, with the
glutamine analog 6-diazo-5-oxonorleucine (DON), protected fat cells
against glucose-induced insulin resistance (13). As shown in Fig.
5A treatment of muscles with
DON was effective in inhibiting GFAT, as evidenced by a large decrease
in the concentration of UDP-N-acetylhexosamines. However,
despite the marked decrease in UDP-HexNAcs, DON had no protective
effect against the development of glucose-induced insulin resistance
(Fig. 5B).
Inhibition of mRNA and Protein Synthesis Protects against
Glucose-induced Insulin Resistance--
In their experiments on
primary cultures of adipocytes, Marshall et al. (14) found
that the inhibitors of mRNA synthesis, actinomycin D and
5,6-dichloro-1- Effect of Protein Kinase C Inhibitor Calphostin C--
Activation
of protein kinase C (PKC) has been reported to cause insulin resistance
by means of serine phosphorylation of the insulin receptor (21, 24).
Furthermore, the insulin resistance induced by a high glucose
concentration has been attributed to PKC activation (5, 21, 22).
Therefore, to evaluate the role of PKC in glucose-induced insulin
resistance in our muscle preparation, we examined the effect of the PKC
inhibitor calphostin C, which acts at the diacylglycerol and phorbol
ester binding site (43). Calphostin C, at a concentration (0.5 µM) that has an inhibitory effect on phorbol
ester-stimulated PKC activity in our muscle preparation (44), did not
protect against the decrease in insulin responsiveness induced by high
glucose and insulin (Fig. 7).
Insulin Signaling--
Activation of PI 3-kinase by insulin is
usually measured after brief exposure of muscle to a high insulin
concentration. This approach was not possible in the present study as
we were studying the effects of prolonged exposure of muscle to high
concentrations of glucose and insulin. We therefore measured PI
3-kinase activity in muscles subjected to the same treatment as in the
experiments in which 3MG transport was measured, i.e. after
incubation with 2 milliunits/ml insulin and either 5 or 36 mM glucose for 5 h. As shown in Fig.
8, phosphotyrosine-associated PI 3-kinase
activity was increased ~3-fold in muscles treated with insulin. There
was no significant difference in insulin-stimulated PI 3-kinase
activity between muscles incubated with 5 or 36 mM
glucose.
Phosphorylation of PKB on Ser473 and Thr308 was
also determined in muscles that were incubated with glucose and insulin
for 5 h. Phosphorylated PKB, which was too low to quantify in
muscles incubated without insulin, increased to the same extent in
response to insulin in the muscles incubated with 5 or 36 mM glucose (Fig. 9).
Does Glucose-6-Phosphate Mediate the Effect of High
Glucose?--
In liver and fat cells, rapid influx of glucose induces
an increase in the expression of a number of enzymes involved in
glucose and fat metabolism (45). It has been proposed that
glucose-6-phosphate provides the signal for the increase in enzyme
transcription; this hypothesis is based on the finding that
2-deoxy-D-glucose-6-phosphate (2DG-6-P) mimics the
effect of high glucose on fatty acid synthase expression in adipocytes
(46). In this context we determined whether 2DG-6-P accumulation
decreases insulin responsiveness of glucose transport in muscle.
Muscles were incubated for 5 h with 4 mM pyruvate as
substrate, 2 milliunits/ml insulin, and 8 mM 2DG for the
first 30 min and with 0.5 mM 2DG for the last 4.5 h.
This procedure resulted in the accumulation of 5.4 ± 0.78 µmol/g 2DG-6-P after 30 min and 5.74 ± 1.07 µmol/g 2DG-6-P
after 5 h (means ± S.E. for 8 muscles/muscle group) but had
no effect on maximally insulin-stimulated 3MG transport (1.21 ± 0.13 µmol·ml Protection against Glucose-induced Insulin Resistance by
AICAR--
In liver and fat cells, the rapid influx of glucose induces
a number of enzymes involved in glucose and fat metabolism within ~4
h (45-48). There is evidence suggesting that the dephosphorylation of
transcription factor(s) by protein phosphatases 1 and/or 2A is involved
in this effect, which is prevented by okadaic acid or calyculin (46,
47). We could not use okadaic acid to evaluate whether a similar
mechanism mediates glucose-induced insulin resistance in muscle,
because it causes a marked inhibition of insulin-stimulated glucose
transport, which does not reverse in the time frame of our experiments.
It has also been shown that various kinases (45-48), including
AMP-activated protein kinase (AMPK) (48), prevent activation of gene
transcription by glucose in hepatocytes, presumably by keeping the
transcription factor(s) in the phosphorylated, inactive state.
5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR) enters cells and
is converted to the AMP analog ZMP
(5-aminoimidaazole-4-carboxamide-1- Previous studies have shown that high muscle glycogen
concentrations are associated with decreased responsiveness of glucose transport to insulin (7, 9, 11, 18) and that low glycogen levels are
associated with enhanced insulin action (52, 53). The smaller increase
in glucose transport in response to insulin in muscles that have a high
glycogen content is due to translocation of fewer GLUT4 to the
cell surface (18). It has been hypothesized that the insulin resistance
of glucose transport associated with a high glycogen concentration is
mediated by the association of GLUT4-containing vesicles with the
glycogen-protein complex, making them unavailable for translocation to
the cell surface (19, 20). Despite considerable investigation,
experimental support for this attractive hypothesis has remained
elusive, and our finding that actinomycin D completely protects against
glucose-induced insulin resistance despite a massive increase in
glycogen argues strongly against it. We therefore conclude that a high
muscle glycogen level serves as a marker for rapid influx of glucose into muscle cells but is not the cause of the decrease in insulin action.
There is evidence that a high glucose concentration inhibits insulin
action by inducing a protein kinase C-mediated serine phosphorylation
of the insulin receptor in fibroblasts transfected with the human
insulin receptor (21), and it has been hypothesized that this is the
mechanism that mediates glucose toxicity-induced insulin resistance (5,
21, 22). However, decreased insulin receptor activation, resulting in
impaired insulin signaling, is not the mechanism responsible for the
glucose-induced insulin resistance in our muscle preparation. This is
evidenced by the finding that the stimulation of PI 3-kinase activity
and the phosphorylation of PKB by insulin were not significantly
reduced. Furthermore, the protein kinase C inhibitor calphostin C,
which acts at the diacylglycerol binding site (43), did not protect
against glucose-induced insulin resistance.
Much evidence has accumulated suggesting that hexosamine pathway end
products are responsible for glucose-induced insulin resistance. The
concept that the hexosamine synthetic pathway mediates glucose toxicity
originated from an elegant series of studies by Marshall and co-workers
(13, 14, 30, 54). Their initial studies showed that rapid influx of
glucose, mediated by exposure of cells to high concentrations of both
glucose and insulin, resulted in the development of insulin resistance
of glucose transport (30) but only when glutamine was included in the
incubation medium (54). The finding that glutamine was necessary
suggested the possible involvement of the hexosamine biosynthetic
pathway. Support for this possibility was provided by experiments in
which inhibition of GFAT, the initial and rate-limiting step in the
hexosamine pathway, prevented the development of glucose induced-insulin resistance (13). Further evidence for involvement of
the hexosamine pathway was provided by the finding that glucosamine, which enters the pathway beyond GFAT, is an ~40-fold more potent inducer of insulin resistance than glucose (13). Subsequent studies in
which insulin resistance of glucose transport developed in rats infused
with glucosamine (26, 27) and in muscles incubated with glucosamine
(28, 29) showed that the phenomenon also occurs in skeletal muscle,
supporting the concept that accumulation of UDP-HexNAcs inhibits
insulin stimulation of glucose transport. Further evidence for
involvement of the hexosamine pathway was provided by the finding that
glucose disposal was reduced in transgenic mice overexpressing GFAT in
muscle and adipocytes (55).
In our experiments on isolated muscles, we found that, as in the
studies on fat cells (30), rapid influx of glucose induced by
incubation with high concentrations of glucose and insulin, rather that
just exposure to high glucose, is necessary for the development of
insulin resistance of glucose transport. However, in contrast to the
finding that inclusion of glutamine in the medium was essential for
development of glucose toxicity in fat cells (54), glucose-induced
insulin resistance developed in skeletal muscle despite the absence of
glutamine in the incubation medium. Also, unlike the findings on fat
cells (13), the GFAT inhibitor DON did not protect muscle against
development of glucose toxicity despite prevention of an increase in
UPD-HexNAcs concentration. Marshall et al. (14) found that
inhibition of mRNA synthesis with actinomycin D or DRB completely
protected against glucose-induced insulin resistance in fat cells. They
attributed this protective effect to the inhibition of synthesis of a
protein with a short half-life and hypothesized that the protein might
be GFAT (14). This interpretation was clouded by the puzzling finding
that the protein synthesis inhibitor cycloheximide did not protect
against glucose-induced insulin resistance (15). As in the studies on fat cells, we found that actinomycin D and DRB completely protected against development of insulin resistance in muscles incubated with
high glucose and insulin. However, in contrast to the finding in fat
cells (15), cycloheximide also prevented the decrease in
insulin-stimulated glucose transport in muscle.
These findings are in keeping with the conclusion of Marshall et
al. (14) that inhibition of synthesis of a protein with a short
half-life protects against development of glucose toxicity. However, it
is clear from our results that this protein is not GFAT, as we found
that treatment with cycloheximide or actinomycin D resulted in large
increases in UDP-HexNAcs in muscles incubated with high glucose and
insulin. This finding is probably explained by increased glucose influx
due to prevention of insulin resistance. The findings that
cycloheximide prevents development of insulin resistance despite
markedly increasing UDP-HexNAcs concentration, and that DON does not
protect against glucose toxicity despite lowering UDP-HexNAcs, provides
strong evidence that accumulation of hexosamine pathway end products is
not responsible for glucose-induced insulin resistance in muscle.
How then can the evidence that glucosamine causes insulin resistance
(26, 27, 29) be explained? One hypothesis, proposed by Hresko et
al. (56), on the basis of their findings in fat cells, is that the
insulin resistance induced by glucosamine is caused by depletion of
ATP. Although it is clear from the study by Hresko et al.
(56) and from similar findings obtained by us in isolated
muscle2 that this mechanism
can account for glucosamine-induced insulin resistance under some
experimental conditions, glucosamine can also cause insulin resistance
under conditions that do not result in a decrease in ~P (57).
However, it appears from the findings of Nelson et al. (58)
that glucose and glucosamine induce insulin resistance by different
mechanisms. In any case, our findings provide evidence that conversion
of glucose to glucosamine is not necessary for development of
glucose-induced insulin resistance in muscle, because inhibition of
GFAT does not have a protective effect.
In liver and fat cells, a rapid influx of glucose induces increased
expression of several enzymes involved in glucose and fat metabolism
within ~4 h (45-48). The activity of many transcription factors is
regulated by their phosphorylation state. There is evidence that
protein dephosphorylation is involved in glucose-induced gene
expression (46-48) and that activation of either of two closely related protein kinases, AMPK or
Ca2+/calmodulin-dependent protein kinase,
prevents activation of gene transcription by glucose in hepatocytes
(47, 48). Our finding that AICAR, which activates AMPK (49), protects
against glucose toxicity suggests that dephosphorylation of a
transcription factor may be involved in induction by glucose of a
protein that causes insulin resistance and that activation of AMPK
prevents insulin resistance by keeping the transcription factor in the
phosphorylated state.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP were purchased from PerkinElmer Life
Sciences. 2-Deoxy-D-[1,2-3H]glucose
was purchased from American Radiolabeled Chemicals. Reagents for
SDS-polyacrylamide gel electrophoresis were obtained from Bio-Rad.
Polyclonal antibody F349 against GLUT4 protein was a gift from Dr. Mike
Mueckler of Washington University School of Medicine. All other
reagents were purchased from Sigma Aldrich Chemical Co.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Plasma glucose, insulin, and muscle glycogen levels in glucose- or
saline-infused rats
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Fig. 1.
Effect of 24 h of glucose infusion on
insulin-stimulated muscle 2DG transport. Rats were given glucose
by intravenous infusion to raise plasma glucose and insulin
(black bars). Controls were infused with saline (white
bars). After 24 h of glucose or saline infusion, rats were
anesthetized and epitrochlearis muscles were excised. The muscles were
incubated for 60 min in the absence (Basal) or presence of
insulin, either 60 microunits/ml or 2 milliunits/ml. Muscles were then
washed in glucose-free medium for 10 min followed by measurement of
glucose transport activity using 2DG as described under "Experimental
Procedures." Values were the means ± S.E. for 6-8
muscles/group. Black bars, saline-infused; white
bars, glucose-infused. *, p < 0.05; **,
p < 0.01; glucose-infused versus
saline-infused.
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Fig. 2.
Increase in GLUT4 in plasma membrane.
Rats infused for 24 h with glucose or saline were anesthetized and
given an intravenous infusion of insulin for 30 min to raise plasma
insulin concentration above 2.5 milliunits/ml. The
gastrocnemius/plantaris muscle group was then excised and used for
preparation of a plasma membrane fraction, which was used to measure
GLUT4 content (see "Experimental Procedures"). A representative
Western blot of GLUT4 in the plasma membrane fraction is shown at the
top of the figure. Filled-in portions
of bars represent the increase in GLUT4 in response to
insulin. Each bar represents the mean ± S.E. for
muscles from 6 rats. *, p < 0.05, glucose-infused
versus saline-infused.
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Fig. 3.
Insulin responsiveness of muscles exposed to
different concentrations of glucose for 5 h. Epitrochlearis
muscles were incubated for 5 h in the absence (Basal)
or presence of insulin (2 milliunits/ml) for 5 h with different
concentrations of glucose. Muscles were then washed for two 10-min
periods in glucose-free medium followed by measurement of 3MG transport
as described under "Experimental Procedures." When insulin was
present during the initial incubation it was also present during the
washes and 3MG transport measurement. Values are the means ± S.E.
for 12-25 muscles/group.
Comparison of 3MG transport rates and glycogen concentrations in
muscles incubated for 1 or 5 h
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Fig. 4.
Muscles exposed to 36 mM glucose
without insulin do not become insulin-resistant. Muscles were
incubated with either 5 mM (Control) or 36 mM (High Glucose) glucose for 5 h with
insulin, 2 milliunits/ml (black bars), or without insulin
(white bars). All muscles were then washed for two 10-min
periods in glucose-free medium containing 2 milliunits/ml insulin
followed by measurement of 3MG transport in medium containing 2 milliunits/ml insulin as described under "Experimental Procedures."
Values are the means ± S.E. for 8-10 muscles/group. *,
p < 0.001 versus other groups.
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Fig. 5.
Effects of inhibition of GFAT and of mRNA
and protein synthesis on UDP-HexNAc accumulation and insulin resistance
in muscles exposed to high glucose. After a 60-min preincubation
with or without 200 µM DON, 75 µM
cycloheximide (CYH), or 2 µM actinomycin D
(AD), muscles were incubated for 5 h with 5 mM (Control) or 36 mM (High
Glucose) glucose, 2 milliunits/ml insulin and, when present in the
prior incubation, DON, cycloheximide, or actimomycin D. A,
some muscles were then frozen and used for measurement of UDP-GlcNAc
and UDP-GalNAc. Each bar represents the mean ± S.E.
for the sum of UDP-GlcNAc and UDP-GalNAc (UDP-HexNAcs) for 5 or 6 sets
of pooled muscles. *, p < 0.001 for high glucose with
no inhibitor versus control and high glucose plus DON;
+, p < 0.001 versus high
glucose with no inhibitor. B, other muscles were then washed
in glucose-free medium to remove glucose from the extracellular space
followed by measurement of 3MG transport. Each bar
represents the mean ± S.E. for 7-14 muscles. *,
p < 0.001 versus control groups and
cycloheximide and actinomycin, high glucose groups.
-D ribofuranosylbenzimidazole (DRB), prevented
glucose-induced insulin resistance. They attributed this protective
effect to inhibition of the synthesis of a short-lived protein, which
they thought was probably GFAT. We found that both actinomycin D (Fig.
5B) and DRB (data not shown) also completely prevent the
insulin resistance induced by high glucose and insulin in skeletal
muscle. Cycloheximide, at a concentration that inhibits protein
synthesis in our muscle preparation (32), has a similar protective
effect (Fig. 5B). None of these inhibitors had any effect on
insulin-stimulated glucose transport in control muscles incubated with
5 mM glucose (Fig. 5B). They also had no effect on basal transport, and in the case of cycloheximide, basal 3MG transport averaged 0.203 ± 0.05 µmol·ml
1·10
min
1 in muscles incubated for 5 h with 5 mM glucose and 0.217 ± 0.02 µmol·ml
1·10
min
1 for muscles incubated for 5 h with
glucose and 75 µM cycloheximide (means ± S.E. for 6 muscles/group). The prevention of glucose-induced insulin resistance by
inhibition of protein synthesis is clearly not mediated by a decrease
in GFAT in muscle; this is evident from the finding that UDP-HexNAc
concentrations were markedly higher in the muscles treated with
cycloheximide or actinomycin D (Fig. 5A). This greater
increase in UDP-HexNAcs is likely due to more glucose being transported
into the muscle cells and entering the hexosamine synthetic pathway
because of the prevention of insulin resistance. Similarly, glycogen
accumulation was increased in muscles in which insulin resistance was
prevented by actinomycin D or cycloheximide (Fig.
6). This effect on glycogen was greater with actinomycin D, possibly because of an inhibitory effect of cycloheximide on the activation of glycogen synthase by insulin (42).
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Fig. 6.
Glycogen accumulation is increased in muscles
incubated with actinomycin D or cycloheximide. Muscles were
incubated with or without 2 µM actinomycin D
(AD) or 75 µM cycloheximide (CYH),
2 milliunits/ml insulin, and 5 (Control) or 36 mM glucose using the same protocol as described in Fig. 5
except that instead of measurement of 3MG transport, muscles were
clamp-frozen and used for measurement of glycogen. Values are the
means ± S.E. for 12-18 muscles. *, p < 0.05;
**, p < 0.001, versus high glucose with no
inhibitor.
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Fig. 7.
The protein kinase C inhibitor calphostin C
does not protect against muscle insulin resistance. Muscles were
incubated for 5 h with 36 mM glucose and 2 milliunits/ml insulin with or without 0.5 µM calphostin
C. Control muscles were incubated for 5 h with 5 mM
glucose and 2 milliunits of insulin. Muscles were then washed with
glucose-free medium followed by measurement of 3MG transport.
Each bar represents the mean ± S.E. for 7 muscles. *, p < 0.001 versus control
group.
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[in a new window]
Fig. 8.
Phosphotyrosine-associated PI 3-kinase
activity is similar in muscles exposed to 5 or 36 mM
glucose. Muscles were incubated in the absence (white
bar) or presence (black bars) of insulin (2 milliunits/ml) and 5 mM (Control) or 36 mM glucose for 5 h and then clamp-frozen. Muscles
extracts were used to prepare phosphotyrosine immunoprecipitates, which
were assayed for PI 3-kinase activity as described under
"Experimental Procedures." A representative autoradiogram showing
32P incorporation into phosphatidylinositol is shown at the
top of the figure. The average of the optical density values
for the basal samples was set at 100. Values for the insulin-stimulated
samples were then calculated relative to basal. Values are means ± S.E. for 8-9 muscles.
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Fig. 9.
Exposure of muscles to 36 mM
glucose has no effect on insulin stimulation of PKB
phosphorylation. Muscles were incubated in the absence or presence
of insulin (2 milliunits/ml) and 5 mM (Control)
or 36 mM (High Glucose) glucose for 5 h.
Muscle extracts were resolved by SDS-polyacrylamide gel electrophoresis
(10% gels), transferred to nitrocellulose membranes, and immunoblotted
with antibodies against phospho-PKB Ser473 and
Thr308 as described under "Experimental Procedures." A
representative blot is shown at the top of the figure. The
phospho-Ser473 and phospho-Thr308 bands were
quantified by densitometry. PKB phosphorylation in the absence of
insulin was too low to detect. The average value for the 5 mM glucose group was set at 100, and the values for the 36 mM group were expressed as percentages of the average for
the muscles exposed to 5 mM glucose. There were 12 muscles/group.
1·10
min
1 for muscles incubated with 2DG and
1.26 ± 0.11 µmol·ml
1·10
min
1 for control muscles (n = 8/group)). The concentration of 2DG-6-P attained in the muscles
incubated with 2DG (5.74 ± 1.07 µmol/g) was much higher than
that of glucose-6-phosphate (0.14 ± 0.03 µmol/g;
n = 4) in muscles incubated with 36 mM
glucose and 2 milliunits/ml insulin. These findings do not, of course,
rule out the possibility that glucose-6-phosphate plays a role in
inducing glucose toxicity by a mechanism that is not mimicked by
2DG-6-P.
-D-ribofuranosyl-5'-monophosphate) resulting in the activation of AMPK (49). In this context, we evaluated
the possibility that AICAR might prevent the decrease in
insulin-stimulated glucose transport. A complication with the use of
this approach is that activation of AMPK by AICAR results in
stimulation of glucose transport, and this effect is additive to that
of a maximal insulin stimulus (50, 51). We therefore included AICAR in
the incubation medium for only the first 3 h of the 5-h incubation
to allow its effect on glucose transport to wear off. As shown in Fig.
10, this strategy was successful, as
there was no significant difference in 3MG transport between the 5 mM glucose-exposed muscles incubated with or without AICAR. Treatment with AICAR for the first 3 h of a 5-h incubation with 36 mM glucose and insulin protected muscles against the
glucose-induced decrease in insulin responsiveness of glucose transport
(Fig. 10).
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Fig. 10.
The activator of AMP kinase, AICAR, protects
against glucose-induced insulin resistance. Muscles were incubated
for 3 h with 2 milliunits/ml insulin and 5 mM glucose
(Control) or 36 mM high glucose with
(black bars) or without (white bars) 500 µM AICAR. The muscles were then transferred to fresh
medium with the same insulin and glucose concentrations but without
AICAR and incubated for another 2 h. Muscles were then washed in
glucose-free medium followed by measurement of 3MG transport. Each
bar represents the mean ± S.E. for 13 muscles. *,
p < 0.01 versus all other groups.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
---|
* This work was supported National Institutes of Health Grant DK18986 and Diabetes Research and Training Center Grant DK20579.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported initially by a Nakatomi Health Science Foundation
(Tokyo) postdoctoral fellowship and subsequently by an American Diabetes Association mentor-based postdoctoral fellowship.
§ To whom correspondence should be addressed: Washington University School of Medicine, Div. of Geriatrics and Gerontology, 4566 Scott Ave., Campus Box 8113, St. Louis, MO 63110. Tel.: 314-362-3506; Fax: 314-362-7657; E-mail: jhollosz@im.wustl.edu.
Published, JBC Papers in Press, March 27, 2001, DOI 10.1074/jbc.M010599200
2 D.-H. Han and J. O. Holloszy, unpublished observation.
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ABBREVIATIONS |
---|
The abbreviations used are:
PKB, protein kinase
B;
UDP-HexNAcs, UDP-N-acetylhexosamines
(UDP-N-acetylglucosamine plus
UDP-N-acetylgalactosamine);
GFAT, glutamine:fructose-6-phosphate amidotransferase;
KHB, Krebs-Henseleit
bicarbonate buffer;
2DG, 2-deoxy-D-glucose;
2DG-6-P, 2-deoxy-D-glucose-6-phosphate;
3MG, 3-O-methyl-D-glucose;
DON, 6-diazo-5-oxonorleucine;
DRB, 5,6-dichloro-1--D
ribofuranosylbenzimidazole;
PKC, protein kinase C;
PI 3-kinase, phosphatidylinositol 3-kinase;
AICAR, 5-aminoimidazole-4-carboxamide
ribonucleoside;
AMPK, AMP-activated protein kinase;
TBST, Tris-buffered
saline containing 0.1% Tween 10.
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