Sustained Exposure of L6 Myotubes to High Glucose

and Insulin Decreases Insulin-Stimulated GLUT4

Translocation but Upregulates GLUT4 Activity
Carol Huang,1,2 Romel Somwar,1,3 Nish Patel,1,4 Wenyan Niu,1 Dóra Török,1 and Amira Klip1,3

Hyperglycemia and hyperinsulinemia are cardinal fea- tempts have been made in adipose cells in culture, to
tures of acquired insulin resistance. In adipose cell dissect out the mechanisms leading to this impaired insu-
cultures, high glucose and insulin cause insulin resis- lin response of target tissue, using a high glucose and
tance of glucose uptake, but because of altered GLUT4 insulin model. In 3T3-L1 adipocytes and primary cultured
expression and contribution of GLUT1 to glucose up- rat adipocytes, 24- to 48-h pretreatment with high glucose
take, the basis of insulin resistance could not be ascer- and insulin resulted in decreases in insulin-stimulated
tained. Here we show that GLUT4 determines glucose glucose uptake, GLUT4 translocation to the cell surface,
uptake in L6 myotubes stably overexpressing myc-
tagged GLUT4. Preincubation for 24 h with high glucose insulin receptor substrate (IRS)-1 tyrosine phosphoryla-
and insulin (high Glc/Ins) reduced insulin-stimulated tion, and phosphatidylinositol (PI) 3-kinase activity (3– 6).
GLUT4 translocation by 50%, without affecting GLUT4 It is difficult to ascribe the insulin resistance to a single
expression. Insulin receptor and insulin receptor sub- mechanism, because GLUT4 levels dropped in some of
strate-1 tyrosine phosphorylation, phosphatidylinositol those studies, and GLUT1 also contributes to glucose
3-kinase activation, and Akt phosphorylation also di- uptake in 3T3-L1 adipocytes.
minished, as did insulin-mediated glucose uptake. How- Although GLUT4 levels diminish in adipose cells of
ever, basal glucose uptake rose by 40% without any gain diabetic animals and humans, they are not altered in
in surface GLUT4. High Glc/Ins elevated basal p38 mi-
togen-activated protein kinase (MAPK) phosphoryla- skeletal muscle (7–9). Therefore, the mechanisms leading
tion and activity, and a short inhibition of p38 MAPK to acquired insulin resistance may differ in muscle and fat
with SB202190 corrected the rise in basal glucose up- tissues. To date, there are no studies of acquired insulin
take, suggesting that p38 MAPK activity contributes to resistance in skeletal muscle cells that analyze the differ-
this rise. We propose that in a cellular model of skeletal ent steps governing glucose uptake. The objective of the
muscle, chronic exposure to high Glc/Ins reduced the present study was to analyze the effect of high glucose and
acute, insulin-elicited GLUT4 translocation. In addition, insulin on GLUT4 translocation, glucose uptake, and the
basal state GLUT4 activity was augmented to partially insulin signaling pathway, in a muscle cell line where
compensate for the translocation defect, resulting in a
more robust glucose uptake than what would be pre- GLUT4 is the predominant transporter. L6 myotubes over-
dicted from the amount of cell surface GLUT4 alone. expressing myc-tagged GLUT4 offer these possibilities, as
Diabetes 51:2090 –2098, 2002 well as afford the opportunity to compare GLUT4 translo-
cation with glucose uptake in intact cell preparations, as
required to assess possible changes in GLUT4 activity.
Indeed, several studies suggest that GLUT4 activity may be

I
n type 2 diabetes, there is a failure to increase regulated (10 –15). Depending on the experimental tech-
glucose disposal into peripheral tissues in response nique used to measure GLUT4 translocation, the fold
to insulin, leading to chronically elevated levels of increase in translocation matches to different extents the
glucose in the circulation followed by a compensa- fold increase in glucose uptake (10 –12). In addition,
tory rise in insulin (1). The elevated glucose and insulin several conditions lead to a dissociation of GLUT4 trans-
levels in turn exacerbate insulin resistance, contributing location from stimulation of glucose uptake (13–15). Re-
significantly to the pathogenesis of the disease (2). At- cently, we demonstrated that SB203580, a selective
inhibitor of p38 mitogen-activated protein kinase (MAPK)
(16), decreased insulin-stimulated glucose uptake in rat
From the 1Programme in Cell Biology, Hospital for Sick Children, Toronto,
Canada; the 2Institute of Medical Science, University of Toronto, Toronto, skeletal muscle, L6 myotubes, and 3T3-L1 adipocytes
Canada; the 3Department of Biochemistry, University of Toronto, Toronto, without reducing GLUT4 translocation (17,18), raising the
Canada; and the 4Department of Physiology, University of Toronto, Toronto, possibility that p38 MAPK can regulate the intrinsic activ-
Canada.
Address correspondence and reprint requests to Amira Klip, Programme in ity of GLUT4. It is currently unknown whether GLUT4
Cell Biology, The Hospital for Sick Children, 555 University Ave., Toronto, ON, activity or p38 MAPK is altered in insulin resistance. We
Canada M5G 1X8. E-mail: amira@sickkids.ca.
Received for publication 16 October 2001 and accepted in revised form 3
report that 24-h pretreatment with 25 mmol/l glucose and
April 2002. 100 nmol/l insulin (high Glc/Ins) of L6-GLUT4myc myo-
ATF, activating transcription factor; Glc/Ins, glucose/insulin; IC50, half- tubes activates p38 MAPK and stimulates basal-state glu-
maximal inhibitory concentration; IR, insulin receptor; IRS, insulin receptor
substrate; JNK, Jun NH2-terminal kinase; MAPK, mitogen-activated protein cose uptake mediated by GLUT4myc, without increasing
kinase; OPD, o-phenylenediamine dihydrochloride; PI, phosphatidylinositol. cell surface GLUT4myc levels. The pretreatment also
2090 DIABETES, VOL. 51, JULY 2002
 C. HUANG AND ASSOCIATES

causes a reduction in insulin-dependent GLUT4myc trans- resolving the protein by 7.5% SDS-PAGE. IRS-1 was immunoprecipitated from
300 ␮g protein, and the immunoprecipitates were resolved by 7.5% SDS-PAGE.
location and in the IRS-1–phosphatidylinositol 3-kinase– Tyrosine phosphorylation of IR and IRS-1 and the association of p85 with
Akt pathway. The elevation in basal glucose uptake may IRS-1 were determined by immunoblotting with anti-phosphotyrosine or
be an adaptive change developed to counteract the defect anti-p85 antibodies at 1:2,000, 1:1,000, and 1:1,000 dilutions, respectively. Total
in GLUT4 translocation. IRS-1 content was determined in whole cell lysates, prepared by the same
method as described above for detection of total cellular p38 MAPK.
PI 3-kinase activity assay. PI 3-kinase activity associated with phosphoty-
RESEARCH DESIGN AND METHODS rosine immunoprecipitates was measured in vitro toward phosphatidylinositol
Materials. Anti-GLUT1 and anti-GLUT4 antisera were raised in rabbits to as described previously (24)
peptides encompassing 12 COOH-terminal residues of each protein (19); Statistical analysis. Statistical analysis was performed using either unpaired
anti-GLUT3 was generated using the 19 COOH-terminal peptides (20). Anti- Student’s t test or ANOVA (Fisher, multiple comparisons), as applicable.
p38 MAPK, anti-insulin receptor ␤ (C-19), anti-myc (9E10) antibodies, and
anti-p85 subunit antibodies were purchased from Santa Cruz Biotechnology
(Santa Cruz, CA). Phosphospecific antibodies to p38 MAPK, Jun NH2-terminal
kinase (JNK), and activating transcription factor (ATF)-2 fusion protein were RESULTS
from New England Biolabs (Mississauga, ON, Canada). Anti-phosphotyrosine Exposure of L6-GLUT4myc myotubes to high Glc/Ins
and anti–IRS-1 antibodies were from Upstate Biotechnology (Lake Placid, increased basal glucose uptake but decreased the
NY). o-Phenylenediamine dihydrochloride (OPD reagent) was from Sigma.
SB202190 and blasticidin-HCl were from Calbiochem (La Jolla, CA). Indinavir
acute insulin-mediated glucose uptake. We first exam-
was a gift from Dr. Ralph Germinario (Sir Mortimer B. Davis Jewish General ined whether muscle cells were susceptible to the effect of
Hospital, Montreal, Canada) and Dr. Nava Bashan (Ben-Gurian University, preincubation with high glucose and insulin. L6-
Béer-Sheva, Israel). GLUT4myc myotubes were incubated for 24 h with 25
L6-GLUT1myc cells, L6-GLUT4myc cells, cell culture, and incubations.
L6 muscle cells stably expressing myc-tagged GLUT4myc (L6-GLUT4myc
mmol/l glucose and 100 nmol/l insulin and then for 5 h in
cells) were described previously (21,22). L6 stably expressing GLUT1myc serum-free medium (5 mmol/l glucose). Subsequently, the
were prepared by cotransfection of L6 myoblasts with pCX2-GLUT1myc and rates of hexose uptake in the basal state and after an acute
pSV2-bsr, a blasticidin S deaminase expression plasmid, and selected with (30-min) insulin challenge were determined (Fig. 1A). High
blasticidin-HCl. Glass cloning cylinders (Bellco Glass, Vineland, NJ) were Glc/Ins pretreatment increased basal 2-deoxyglucose up-
used to select individual colonies for expansion. Each clonal line was
maintained in 2 ␮g/ml blasticidin-HCl and tested for GLUT1myc expression
take by 40% (control, 25.2 ⫾ 1.7; high Glc/Ins, 34.9 ⫾ 1.3
and the ability to differentiate into myotubes. GLUT1 or GLUT4-myc myo- pmol 䡠 min⫺1 䡠 mg⫺1 protein; P ⬍ 0.005). In control cells,
blasts were differentiated into myotubes, pretreated with 25 mmol/l glucose acute insulin stimulation elevated 2-deoxyglucose uptake
and 100 nmol/l insulin (high Glc/Ins) for 24 h, and deprived of serum for 5 h by 2.3-fold (basal, 25.2 ⫾ 1.7; insulin, 58.7 ⫾ 4.9 pmol 䡠
in 5 mmol/l glucose before incubation with inhibitors, followed by acute
min⫺1 䡠 mg⫺1 protein; ⌬, 33.5 pmol 䡠 min⫺1 䡠 mg⫺1 protein;
insulin challenge. Inhibitors were administered in DMSO, with maximum
concentration of 0.1% vol/vol in the incubation medium and without effect on P ⫽ 0.0002). In contrast, myotubes that were pretreated
the parameters measured. with high Glc/Ins for 24 h had a significantly smaller
Determination of 2-deoxyglucose uptake. 2-Deoxyglucose uptake mea- response to the acute insulin challenge, showing only a
surements were carried out as described previously (18) for 5 min in 1.6-fold increase in 2-deoxyglucose uptake (basal, 34.9 ⫾
HEPES-buffered saline containing 10 ␮mol/l 2-[3H]deoxyglucose (0.5 ␮Ci/ml)
in the absence of insulin and inhibitors, unless otherwise indicated. Nonspe-
1.3; insulin, 56.9 ⫾ 3.1 pmol 䡠 min⫺1 䡠 mg⫺1 protein; ⌬, 22.0
cific uptake was determined in the presence of 10 ␮mol/l cytochalasin B and pmol 䡠 min⫺1 䡠 mg⫺1 protein; P ⫽ 0.0002).
subtracted from all experimental values. Exposure of L6-GLUT4myc myotubes to high Glc/Ins
Measurement of GLUT4myc translocation in L6 myotubes. The amount decreased the acute insulin-mediated GLUT4 trans-
of myc-tagged GLUT4 at the surface of intact cells was measured by an
location. To elucidate the mechanism by which high
antibody-coupled colorimetric assay as validated previously (23). Briefly,
monolayer of myotubes were exposed to anti-myc antibody (1:100) for 60 min, Glc/Ins pretreatment altered glucose uptake, we measured
fixed with 4% paraformaldehyde for 10 min, and then incubated with peroxi- surface GLUT4 levels under the various treatments. L6-
dase-conjugated donkey anti-mouse IgG (1:1,000) for 30 min, all at 4°C. Cells GLUT4myc cells stably express GLUT4 tagged with an
were washed extensively, and 1 ml of OPD reagent was added for 30 min at exofacial myc epitope (GLUT4myc). GLUT4myc segre-
room temperature. The reaction was stopped by 0.25 ml of 3N HCl. The
supernatant was collected, and the absorbance was measured at 492 nm.
gates, cycles, and responds to insulin in a manner similar
Nonspecific IgG binding, as measured by a peroxidase-conjugated anti-mouse to endogenous GLUT4 (21–23). The amount of GLUT4myc
IgG, was subtracted from experimental values. incorporated into the plasma membrane was quantitated
Detection of total cellular p38 MAPK, protein phosphorylation, and by immunologically labeling the myc epitope at the surface
assay of p38 MAPK activity. Total p38 MAPK protein and p38 MAPK
of intact cells. Acute insulin stimulation of untreated cells
phosphorylation and activity were measured as previously described (18) with
the following modifications. For p38 MAPK activity, monoclonal mouse increased the amount of GLUT4myc at the cell surface by
anti–phospho-p38 MAPK antibody covalently linked to Sepharose beads (New 2.3 ⫾ 0.2-fold above basal (P ⫽ 0.01) (Fig. 1B). Preincu-
England Biolab) (20 ␮l/condition) was used to immunoprecipitate activated bation of cells for 24 h with high Glc/Ins caused a 45%
p38 MAPK from 200 ␮g total cell lysate. The complex was washed four times reduction in acute insulin-induced recruitment of
with 1 ml wash buffer and twice with 1 ml kinase buffer and then incubated
under constant agitation for 30 min at 30°C with 30 ␮l reaction mixture
GLUT4myc to the plasma membrane (basal, 0.9 ⫾ 0.1;
(kinase buffer containing 200 ␮mol/l ATP and 2 ␮g recombinant ATF-2). insulin, 1.7 ⫾ 0.17; P ⬍ 0.005). However, high Glc/Ins
Reaction was stopped by 50 ␮l of 2⫻ Laemmli sample buffer, and supernatant treatment did not alter surface GLUT4myc under basal
was resolved by 10% SDS-PAGE. Phospho-ATF-2 was determined by immu- conditions (Fig. 1B), despite increasing 2-deoxyglucose
noblotting with anti–phospho-ATF-2 antibody at 1:500 dilution.
Detection of total cellular GLUT1, GLUT3, and GLUT4 proteins. L6-
uptake by 40% (Fig. 1A).
GLUT4myc myoblasts were differentiated into myotubes in 10-cm plates, and Total cellular content of GLUT1 but not GLUT4 was
total cellular membranes were prepared as described previously (24). Poly- increased by chronic pretreatment with high Glc/Ins.
clonal primary antibody was used at 1:2,000 dilution. In Fig. 1, we show that high Glc/Ins pretreatment caused a
Detection of insulin receptor and IRS-1 expression and phosphoryla- significant elevation of basal glucose uptake without in-
tion and IRS-1–associated p85. Acute insulin stimulation (100 nmol/l for 5
min at 37°C) was followed by cell lysis, and whole-cell extracts were prepared
creasing GLUT4myc translocation. These results suggest
from myotubes as described (18,25). Insulin receptor (IR) was immunopre- that GLUT4 may be hyperactive under these conditions or
cipitated from 500 ␮g protein, and the sample was boiled for 3 min before that upregulation of another transporter may account for
DIABETES, VOL. 51, JULY 2002 2091
 INSULIN RESISTANCE AND GLUT4 ACTIVITY

FIG. 2. In L6-GLUT4myc myotubes, GLUT4 is the predominant glucose

transporter. A: L6-GLUT4myc myotubes were incubated in high Glc/Ins.
Total cellular membranes were prepared, and 10 ␮g protein was
immunoblotted for GLUT1 or GLUT4. A representative immunoblot of
three separate experiments is shown. The levels of GLUT1 and GLUT4
cannot be directly compared because of the differences in antibody
immunoreactivity. B: Total cellular membranes were prepared from
3T3-L1 adipocytes, mouse skeletal muscle, L6-GLUT4myc myoblasts
(MB), L6-GLUT4myc myotubes (MT), and L6 parental myotubes (WT).
Protein (5, 10, 25, or 100 ␮g, as indicated) was immunoblotted for
GLUT4. The difference in amount of protein loaded was necessary to
allow for quantitative comparison of GLUT4 in the parental L6 myo-
tubes with GLUT4myc in the transfected cell line on the same SDS-
PAGE. Using the FluorChem 8000 imaging system (Alpha Innotech),
the optical density of each chemiluminescent protein band was mea-
sured within the linear range. The optical density relative to that
obtained for L6 wild-type myocytes was then adjusted according to the
amount of protein loaded on the gel to give the molar ratios of GLUT4
listed below the gel images. The lesser migration of GLUT4 in 3T3-L1
adipocytes in comparison to myocytes (26) and that of myoblasts in
comparison to myotubes (27) have been previously reported.

level of GLUT4myc to that of the endogenous GLUT1. In

parental L6 myotubes, the molar ratio of surface GLUT1 to
GLUT4 is 1.0:0.8, as determined by quantitative photola-
beling using azi-trifluroethylbenzoyl-bis-mannose-yloxypro-
pylamine (ATB-BMPA) (28). In L6-GLUT4myc myotubes,
the myc-tagged transporter is significantly overexpressed
relative to the endogenous GLUT4. Figure 2B shows the
molar ratio of GLUT4myc to that of GLUT4 in other cells
and tissues expressing this transporter. GLUT1 expression
is the same in GLUT4myc cells as in parental L6 cells.
Thus, in L6-GLUT4myc cells, the ratio of GLUT4myc to
GLUT4 to GLUT1 is 124:1:1.2. Hence, GLUT4myc is vastly
the predominant glucose transporter in L6-GLUT4myc
myotubes, and it is likely to be the transporter responsible
for the increase in glucose uptake caused by high Glc/Ins
FIG. 1. Effect of chronic exposure of L6-GLUT4myc myotubes to high (Fig. 1A). To further support this hypothesis, we used
Glc/Ins on glucose uptake and GLUT4 translocation. Myotubes were
incubated for 24 h in growth medium supplemented with 25 mmol/l
indinavir, a protease inhibitor that was shown to inhibit
glucose and 100 nmol/l insulin. Cells were depleted of insulin and GLUT4 but not GLUT1 transporters directly (29,30). Figure
serum in medium containing 5 mmol/l glucose for 5 h and then left 3A shows that when indinavir was present during the
untreated or stimulated for 30 min with 100 nmol/l insulin, followed by
assessment of 2-deoxyglucose uptake (A) and cell surface GLUT4myc 2-deoxyglucose transport assay, there was a dose-depen-
levels (B) in intact cells. Nonspecific antibody binding as measured by dent inhibition of 2-deoxyglucose uptake in the
anti-mouse IgG alone was subtracted from all experimental values and GLUT4myc but not in the GLUT1myc myotubes; a similar
then the amount of GLUT4myc at cell surface under each condition was
expressed relative to that of control cells under basal conditions. dose-response curve was observed after the cells were
Results are means ⴞ SE of five experiments in which each condition pretreated with high Glc/Ins. Taken together, these results
was assayed in quadruplicate. *P < 0.005 vs. respective controls. **P <
0.005 vs. control cells. ⌬, difference between control and acute insulin
suggest that GLUT4myc is the predominant glucose trans-
stimulation; 䡺, basal; f, acute insulin (100 nmol/l for 30 min). porter in these cells responsible for glucose influx. More-
over, the high Glc/Ins effect is via GLUT4.
the increased glucose uptake. To assess these possibilities, It was then of interest to explore whether GLUT1
GLUT1 and GLUT4 levels were determined in total mem- activity is similarly regulated in cells where GLUT1 is the
branes isolated from L6-GLUT4myc myotubes pretreated predominant route for glucose uptake. To this end, we
with high Glc/Ins for 24 h. Total GLUT1 content was used L6 muscle cells overexpressing GLUT1myc, where
increased by 2.5 ⫾ 0.5-fold (P ⬍ 0.05) (Fig. 2A). In GLUT1myc expression is controlled by a constitutively
contrast, high Glc/Ins pretreatment did not alter total active cytomegalovirus promoter, and therefore its levels
GLUT4 content (Fig. 2A). are unlikely to change biosynthetically with high Glc/Ins.
GLUT1 does not contribute to the increase in basal High Glc/Ins preincubation did not elevate the basal level
glucose uptake induced by high Glc/Ins. Because of the of glucose uptake in L6-GLUT1myc myotubes (control,
rise in GLUT1 protein, it was important to compare the 20.6; high Glc/Ins, 21.3 pmol 䡠 min⫺1 䡠 mg⫺1 protein) (Fig.
2092 DIABETES, VOL. 51, JULY 2002
 C. HUANG AND ASSOCIATES

FIG. 4. SB202190 restored basal glucose uptake to control levels in

L6-GLUT4myc myotubes pretreated with high Glc/Ins. Cells were incu-
bated for 24 h in growth medium supplemented with 25 mmol/l glucose
and 100 nmol/l insulin and then depleted of serum and insulin for 5 h
and left untreated or treated with 10 ␮mol/l SB202190 for 50 min.
Insulin (100 nmol/l) was added during the last 30 min of this incuba-
tion. 2-Deoxyglucose uptake was determined in the absence of
SB202190. Results are means ⴞ SE of four experiments in which each
condition was assayed in quadruplicate. *P < 0.01 vs. acute insulin
without SB 202190. **P ⴝ 0.05 vs. control. †P ⴝ 0.05 vs. basal high
Glc/Ins pretreatment. ††P ⴝ 0.001 vs. high Glc/Ins followed by acute
insulin but without SB202190. 䡺, basal; f, acute insulin.

Insulin-activated but not basal p38 MAPK phosphor-

FIG. 3. GLUT4 but not GLUT1 mediates the increased basal glucose
uptake in L6-GLUT4myc myotubes induced by high Glc/Ins. A: 2-Deoxy-
ylation is attenuated by preexposure of L6-
glucose uptake was measured in both control and high Glc/Ins–pre- GLUT4myc myotubes to high Glc/Ins. The abrogation
treated L6-GLUT4myc and L6-GLUT1myc cells, with 0 –50 ␮mol/l by SB202190 of the high Glc/Ins–induced elevation in basal
indinavir present during the transport assay only. 2-Deoxyglucose
uptake results are expressed as a percentage of the value obtained in glucose uptake in GLUT4myc myotubes suggested the
the absence of indinavir. There was no statistical difference in the participation of p38 MAPK. Hence, we determined the
results between control and high Glc/Ins–pretreated cells within each effect of high Glc/Ins on p38 MAPK phosphorylation and
cell type. B: L6-GLUT1myc myotubes were pretreated with high Glc/Ins
for 24 h, and 2-deoxyglucose uptake was assessed. Results obtained in kinase activity in these cells. Activation of p38 MAPK by
parallel using L6-GLUT4myc myotubes are presented for comparison. cytokines (31) and insulin (17) correlates with the dual
Results are means ⴞ SE of five experiments in which each condition
was assayed in quadruplicate. *P < 0.005 vs. controls.
phosphorylation of the enzyme on threonine 180 and
tyrosine 182. Using antibodies that recognize p38 MAPK
3B), suggesting that GLUT1 activity cannot be upregulated only when phosphorylated on both of these residues,
by high Glc/Ins. Collectively, these results highlight that pretreatment of myotubes with high Glc/Ins elevated basal
functionally, GLUT4myc is responsible for both basal and p38 MAPK phosphorylation by 80% (P ⬍ 0.05) (Fig. 5A).
acute insulin-stimulated glucose uptake in L6-GLUT4myc The cellular content of p38 MAPK was also elevated by
myotubes, and the basal state activity of GLUT4, but not of 25% by the high Glc/Ins pretreatment (P ⬍ 0.05, Fig. 5B).
GLUT1, increases upon preincubation of myotubes with We then determined the effect of high Glc/Ins treatment
high Glc/Ins. on p38 MAPK protein kinase activity, using an in vitro
SB202190 restored basal glucose uptake in L6- kinase assay. Consistent with the results of p38 MAPK
GLUT4myc myotubes pretreated with high Glc/Ins to phosphorylation, preincubation of cells with high Glc/Ins
control levels. We have previously reported that an acute for 24 h increased basal p38 MAPK activity by 2.3 ⫾
insulin challenge increases the activity of translocated 0.3-fold (P ⬍ 0.05) (Fig. 5C). We also measured phosphor-
GLUT4, an effect that is reduced by pretreating cells with ylation of JNK, another stress-activated MAPK, to deter-
p38 MAPK inhibitors (17). Therefore, we hypothesized that mine if the effects of high Glc/Ins were specific for p38
the activity of p38 MAPK contributes to the elevated basal MAPK. Treatment with high glucose and insulin for 24 h
glucose uptake observed following high Glc/Ins pretreat- did not increase basal JNK phosphorylation (data not
ment. L6-GLUT4myc myotubes that were pretreated for shown).
24 h with high Glc/Ins were incubated with an inhibitor of Chronic pretreatment with high Glc/Ins decreased
p38 MAPK for 20 min before assaying glucose uptake. the acute insulin-mediated tyrosine phosphorylation
Figure 4 shows that SB202190 restored the basal rate of of insulin receptor, IRS-1, phosphotyrosine-associ-
glucose uptake in cells pretreated with high Glc/Ins, ated PI 3-kinase activity, and Akt phosphorylation.
suggesting that p38 MAPK may regulate the basal state We demonstrate in Fig. 1 that pretreatment of myotubes
activity of GLUT4 in L6-GLUT4myc myotubes preincu- with high Glc/Ins reduced subsequent insulin-stimulated
bated with high Glc/Ins. GLUT4 translocation by 45%. This result could arise from
DIABETES, VOL. 51, JULY 2002 2093
 INSULIN RESISTANCE AND GLUT4 ACTIVITY

FIG. 5. High Glc/Ins elevates basal, but attenuates insulin-stimulated, p38MAPK phosphorylation and kinase activity. L6-GLUT4myc myotubes
were incubated for 24 h in growth medium supplemented with 25 mmol/l glucose and 100 nmol/l insulin. Cells were depleted of serum and insulin
for 5 h, then total cell lysates were prepared in Laemmli sample buffer, and 40 ␮g of protein was immunoblotted for phospho-p38 MAPK (A) and
p38 MAPK (B). C: Cell lysate (300 ␮g) was immunoprecipitated with a monoclonal phospho-p38 MAPK antibody, followed by an in vitro kinase
using ATF-2 fusion protein as the substrate. Results are means ⴞ SE of three experiments. *P < 0.05 vs. respective controls. 䡺, basal; f, acute
insulin.

alterations in the signals thought to regulate GLUT4 trans- expected, acute insulin caused a robust phosphorylation
location or from defects in the fusion machinery at the of Akt at Thr 308 and Ser 473 in control cells, which was
plasma membrane. Hence, we analyzed the effect of high reduced by 60% and 50%, respectively, after 24-h preincu-
Glc/Ins on tyrosine phosphorylation of the IR, IRS-1, bation with high Glc/Ins (Fig. 6D). Therefore, starting at
activation of PI 3-kinase activity, and phosphorylation of the level of IR, we observed a downregulation to acute
Akt/protein kinase B. High Glc/Ins pretreatment of L6- insulin effect on IR, IRS-1 phosphorylation, phosphoty-
GLUT4myc myotubes for 24 h led to a 38% and a 35% rosine associated PI 3-kinase activity, and Akt phosphor-
reduction in insulin receptor protein content (as deter- ylation.
mined by immunoprecipitation from cell lysate) and
insulin-stimulated (5 min) tyrosine phosphorylation of
insulin receptor (P ⫽ 0.002 and 0.003), respectively (Fig. DISCUSSION
6A). Insulin stimulation (5 min) increased IRS-1 phosphor- Insulin resistance is a key feature of type 2 diabetes. In
ylation by fourfold in control cells (P ⬍ 0.005) (Fig. 6B), particular, it has been argued that elevated levels of
which was diminished by 65% after 24 h of high Glc/Ins glucose and insulin are a major cause for the development
pretreatment (P ⬍ 0.005). Concomitantly, there was a 50% of secondary insulin resistance, but the molecular mecha-
(P ⬍ 0.005) reduction in total cellular IRS-1 protein (Fig. nisms remain obscure (32). Therefore, establishment of in
6B). High Glc/Ins for 24 h also reduced IRS-1–associated vitro models of high glucose and insulin state has been
p85 by 80% (Fig. 6C). Similarly, PI 3-kinase activity asso- pursued to allow understanding of the molecular basis of
ciated with anti-phosphotyrosine immunoprecipitates was acquired insulin resistance. Previous studies in 3T3-L1
also reduced by 40% in pretreated cells (Fig. 6C). As adipocytes and primary cultured adipocytes have shown
2094 DIABETES, VOL. 51, JULY 2002
 C. HUANG AND ASSOCIATES

FIG. 6. Exposure of L6-GLUT4myc myotubes to high Glc/Ins decreases the acute insulin-mediated tyrosine phosphorylation of IR, IRS-1,
IRS-1–associated p85, phosphotyrosine-associated PI 3-kinase activity, and Akt phosphorylation. L6-GLUT4myc myotubes were incubated for 24 h
in control or high Glc/Ins medium. IR and IRS-1 were immunoprecipitated from 500 and 300 ␮g total cell lysate, respectively, and immunoblotted
for IR and phospho-IR (A) and IRS-1–associated p85 (C). Total cell lysate (40 ␮g) was immunoblotted for IRS-1 (B) or phospho-Akt (D). C: In
vitro phosphotyrosine-associated PI 3-kinase activity was measured in cell lysates as described in RESEARCH DESIGN AND METHODS. Results are
expressed relative to the control cells, assigned a value of 1. Results are the means ⴞ SE of three to four experiments. *P < 0.005 vs. control.
䡺, basal; f, acute insulin (100 nmol/l for 5–10 min).

that prolonged exposure to high concentrations of insulin precipitable insulin receptor after 24 h of high insulin and
and glucose resulted in increased basal glucose uptake glucose pretreatment in L6-GLUT4myc myotubes. The
and a decrease in acute insulin-mediated glucose trans- insulin-stimulated tyrosine phosphorylation of the recep-
port, the latter attributed to reduced insulin-stimulated tor was similarly decreased by 35%. Similar observations
GLUT4 translocation (3– 6,33). However, it remained pos- have been made in insulin-resistant and diabetic humans
sible that the insulin-resistant state may also be associated (34,35). In agreement with Ricort et al. (5), we found that
with diminished GLUT4 activity. high Glc/Ins treatment also resulted in a significant reduc-
To our knowledge, there are no comprehensive ac- tion in insulin-stimulated IRS-1 tyrosine phosphorylation
counts of the basis of acquired insulin resistance in muscle and total IRS-1 content. Reduced IRS-1 tyrosine phosphor-
cells using a high glucose and insulin model, yet muscle ylation has been reported in skeletal muscle of insulin-
tissue is a primary determinant of glycemic control in vivo. resistant Zucker rats (36) and in humans with type 2
The aim of this study was to examine the alterations in diabetes (37,38) and obesity (39). Increased degradation of
signaling pathways regulating GLUT4 activity and GLUT4 IRS-1 under chronic insulin treatment is one mechanism
translocation when a cell culture of muscle origin was underlying insulin resistance (40). In our cellular model, a
exposed to sustained high levels of glucose and insulin. A slight discrepancy between the percentage reduction of
muscle cell line where GLUT4 is the predominant trans- IRS-1 tyrosine phosphorylation (65% reduction) and reduc-
porter, L6 myotubes overexpressing myc-tagged GLUT4, tion of IRS-1 content (50%) was observed. There are two
offers the opportunity to compare GLUT4 translocation possible explanations: first, the reduction in IRS-1 content
with glucose uptake in intact cell preparations, allowing us was compounded with a reduction in IR phosphorylation
to assess possible changes in GLUT4 activity. and therefore activity, leading to a more significant reduc-
High Glc/Ins induces a GLUT4 translocation defect. tion in IRS-1 phosphorylation than expected from the
We demonstrated in this study that 24 h of high glucose decrease in IRS-1 content. Second, there may be a separate
and insulin reduced insulin-stimulated GLUT4 transloca- defect in the insulin signaling pathway such that it im-
tion, with no significant change in cell surface GLUT4 at paired the tyrosine phosphorylation of the remaining
the basal state. This is consistent with previous reports of IRS-1.
50 –100% reduction in GLUT4 translocation in 3T3-L1 adi- With the reduction in IRS-1 content and its phosphory-
pocytes under similar conditions (4 – 6,33). To understand lation after chronic exposure to high Glc/Ins, we predicted
the mechanism of the translocation defect, we examined and observed a reduction in the acute insulin-dependent
IR and IRS-1 phosphorylation, PI 3-kinase activity, and Akt activation of PI 3-kinase. We also observed a 60% reduc-
phosphorylation. We found a 35% reduction in immuno- tion in insulin-mediated Akt phosphorylation at threonine
DIABETES, VOL. 51, JULY 2002 2095
 INSULIN RESISTANCE AND GLUT4 ACTIVITY

308 and serine 473. Therefore, the defect in translocation vates p38 MAPK in L6-GLUT4myc myotubes (17), 3T3-L1
may be explained in part by the alterations in signal adipocytes (18,44,45), rat skeletal muscle (46,47), and
transduction from IR to IRS-1 and then to PI 3-kinase and vascular smooth muscle (48). The precise role of p38
Akt, resulting in reduced GLUT4 translocation. In contrast, MAPK in insulin-stimulated glucose uptake needs further
the reduction in GLUT4 translocation caused by high evaluation. In 3T3-L1 adipocytes, Kayali et al. (44) demon-
insulin and glucose using 3T3-L1 adipocytes was associ- strated that p38 MAPK was activated by insulin (2.7-fold),
ated with reduced levels of total cellular GLUT4 (6,33). and insulin-stimulated glucose uptake was inhibited in a
GLUT4 is the determinant glucose transporter af- dose-dependent manner by SB203580 and SB202190. How-
fected by high Glc/Ins. In L6-GLUT4myc myotubes pre- ever, the half-maximal inhibitory concentration (IC50) for
treated with high Glc/Ins, there was an increase in total inhibition of glucose transport was determined to be ⬎10
cellular GLUT1 content, which could potentially account ␮mol/l, greater than the reported half-maximal stimulatory
for the increase in basal glucose uptake. Indinavir, an HIV concentration (EC50) for inhibition of p38 MAPK (⬃0.5
protease inhibitor, has been associated with insulin resis- ␮mol/l). Therefore, the authors concluded that p38 MAPK
tance in humans and animals (29,30). Using Xenopus is not involved in insulin-stimulated glucose uptake in
oocytes transfected with GLUT4 or GLUT1, Murata et al. 3T3-L1 adipocytes. However, because SB203580 and
(29) showed that indinavir inhibits GLUT4- but not GLUT1- SB202190 reduce insulin stimulation of glucose uptake by
mediated glucose transport, suggesting this as the basis of only 50% even at maximal concentrations, the IC50 for
insulin resistance in vivo. Accordingly, we found profound inhibition of glucose uptake is closer to 1–2 ␮mol/l (17).
inhibition of both basal and insulin-stimulated 2-deoxyglu- This value is very close to the IC50 for inhibition of
cose uptake in GLUT4myc but not in GLUT1myc cells. phosphorylation of targets of p38 MAPK in intact cells
Indinavir effectively inhibited glucose uptake in L6- (17). Therefore, the reduction in glucose uptake by
GLUT4myc cells pretreated with high Glc/Ins, indicating SB202190 and SB203580 closely parallels that for p38
that this pretreatment did not alter the affinity of glucose MAPK activity.
transporters to indinavir. The elevation in basal glucose In the present study, we observed that with high Glc/Ins,
uptake caused by pretreatment with high glucose and basal-state glucose uptake was elevated without a con-
insulin was entirely abolished by indinavir. These results comitant increase in surface levels of GLUT4 and without
pointed to GLUT4myc as the major glucose transporter in contribution of GLUT1. We interpret these results to
L6-GLUT4myc cells, and suggest that the increase in suggest that high Glc/Ins elevated the intrinsic activity of
GLUT1 protein had no significant contribution to the GLUT4. Therefore, we explored whether this elevation
observed increase in basal glucose uptake caused by high might be caused by a regulatory input via p38 MAPK. To
Glc/Ins pretreatment. Because GLUT4myc also overrides this effect, we examined the status of p38 MAPK expres-
the endogenous GLUT4, the results in this study refer to sion and activity in response to high Glc/Ins. This pretreat-
the activity and translocation of GLUT4myc, in both basal ment caused a 25% gain in total cellular p38 MAPK, an 80%
and high glucose/insulin conditions. increase in p38 MAPK phosphorylation, and a 2.3-fold
GLUT4myc activation by high Glc/Ins. An insulin-stim- increase in the kinase activity measured in vitro. More-
ulated increase in glucose uptake is the final outcome of over, when high Glc/Ins–pretreated L6 myotubes were
any changes that affect the insulin signaling pathway. We treated briefly with SB202190, the elevation in basal glu-
found that in L6-GLUT4myc myotubes, 24-h exposure to cose uptake was reversed to control levels. These results
high Glc/Ins resulted in a 40% increase in basal glucose support the hypothesis that in response to the chronic high
uptake accompanied by a diminished net response to an Glc/Ins environment, p38 MAPK is activated and may
acute insulin stimulation. Because the cell surface GLUT4 contribute to the increase in basal glucose uptake.
was not elevated at the basal state in the pretreated cells, The magnitude of increase in p38 MAPK phosphoryla-
the possibility that the intrinsic activity of GLUT4 was tion (85%) was greater than the increase in p38 MAPK
upregulated was explored. content (25%), suggesting that there was also upregulation
An emerging body of literature suggests that GLUT4 of the upstream kinases or downregulation of phospha-
translocation is probably not sufficient to fully account for tases, which would normally dephosphorylate p38 MAPK.
the effect of insulin on glucose uptake (41). Differences are Alterations in the p38 MAPK signal pathway is unlikely to
noted in the time course (17,42) and sensitivity to wort- be a generalized stress response, however, since there was
mannin (13,43) of insulin-dependent GLUT4 translocation no increase in phosphorylation of other known stress
and stimulation of glucose uptake in both 3T3-L1 adipo- response kinases, such as JNK or the extracellular signal-
cytes and GLUT4myc L6 myotubes. In each instance, regulated kinase (ERK) MAPK (results not shown). In
substantial GLUT4 translocation occurred without com- contrast to basal p38 MAPK, high Glc/Ins treatment re-
mensurate increases in glucose uptake. Other studies have duced the ability of insulin to acutely increase p38 MAPK
also alluded to a dissociation between GLUT4 transloca- phosphorylation, uncovering a correlation between re-
tion and the full insulin response of glucose uptake in vivo duced insulin-stimulated p38 MAPK activity and reduced
(14,15). Recently, we and others observed that SB203580, insulin stimulation of glucose uptake in insulin-resistant
a selective inhibitor of p38MAPK (16), diminished insulin- states in muscle cells.
stimulated glucose uptake by 30 – 60% in L6 myotubes (17) In conclusion, the results summarized in Table 1 suggest
and 3T3-L1 adipocytes (18) without reducing GLUT4 trans- the following model of imposed insulin resistance in
location. These studies have led to the suggestion that L6-GLUT4myc myotubes: high Glc/Ins for 24 h caused a
insulin may increase the intrinsic activity of GLUT4 via a defect in acute insulin-mediated IR and IRS-1 phosphory-
p38 MAPK-dependent mechanism. Indeed, insulin acti- lation, PI 3-kinase activity, and Akt phosphorylation, lead-
2096 DIABETES, VOL. 51, JULY 2002
 C. HUANG AND ASSOCIATES

TABLE 1 5. Ricort JM, Tanti JF, Van Obberghen E, Le Marchand-Brustel Y: Alterations

Effects of 24-h exposure of L6 myotubes to high glucose and in insulin signalling pathway induced by prolonged insulin treatment of
insulin on insulin-mediated GLUT4 translocation, glucose up- 3T3–L1 adipocytes. Diabetologia 38:1148 –1156, 1995
take, and activation of the signal networks that regulate these 6. Thomson MJ, Williams MG, Frost SC: Development of insulin resistance in
3T3–L1 adipocytes. J Biol Chem 272:7759 –7764, 1997
processes
7. Garvey WT, Maianu L, Huecksteadt TP, Birnbaum MJ, Molina JM, Ciaraldi
Insulin-stimulated values TP: Pretranslational suppression of a glucose transporter protein causes
(fold above respective insulin resistance in adipocytes from patients with non-insulin- dependent
diabetes mellitus and obesity. J Clin Invest 87:1072–1081, 1991
basal)
8. Garvey WT, Maianu L, Hancock JA, Golichowski AM, Baron A: Gene
Control High Glc/Ins expression of GLUT4 in skeletal muscle from insulin-resistant patients
with obesity, IGT, GDM, and NIDDM. Diabetes 41:465– 475, 1992
Glucose uptake 2.3 1.6 9. Sinha MK, Raineri-Maldonado C, Buchanan C, Pories WJ, Carter-Su C,
p38 MAPK phosphorylation 2.6 1.5 Pilch PF, Caro JF: Adipose tissue glucose transporters in NIDDM: de-
p38 MAPK activity 4.7 1.6 creased levels of muscle/fat isoform. Diabetes 40:472– 477, 1991
Surface GLUT4myc levels 2.3 1.7 10. Lund S, Holman GD, Zierath JR, Rincon J, Nolte LA, Clark AE, Schmitz O,
GLUT4 content* 1 1 Pedersen O, Wallberg-Henriksson H: Effect of insulin on GLUT4 cell
IR content* 1 0.7 surface content and turnover rate in human skeletal muscle as measured
IR phosphorylation 1 0.7 by the exofacial bis-mannose photolabeling technique. Diabetes 46:1965–
IRS-1 phosphorylation 4 1.5 1969, 1997
IRS-1 content* 1 0.5 11. Wilson CM, Cushman SW: Insulin stimulation of glucose transport activity
PI-3 kinase activity 7 4.2 in rat skeletal muscle: increase in cell surface GLUT4 as assessed by
photolabelling. Biochem J 299:755–759, 1994
Akt phosphorylation (T308) 8.5 4
12. Vannucci SJ, Nishimura H, Satoh S, Cushman SW, Holman GD, Simpson
Akt phosphorylation (S473) 6 3 IA: Cell surface accessibility of GLUT4 glucose transporters in insulin-
L6-GLUT4myc myotubes were treated with 25 mmol/l glucose and stimulated rat adipose cells. Modulation by isoprenaline and adenosine.
100 nmol/l insulin (high Glc/Ins) for 24 h, followed by incubation in Biochem J 288:325–330, 1992
serum-free medium containing 5 mmol/l glucose for 5 h. Acute 13. Hausdorff SF, Fingar DC, Morioka K, Garza LA, Whiteman EL, Summers
insulin challenge (100 nmol/l) was then given for 5 min for IRS-1 SA, Birnbaum MJ: Identification of wortmannin-sensitive targets in 3T3–L1
phosphorylation, 10 min for p38 MAPK phosphorylation and activity, adipocytes: dissociation of insulin-stimulated glucose uptake and GLUT4
and 30 min for GLUT4 translocation and glucose uptake assay. translocation. J Biol Chem 274:24677–24684, 1999
*Basal value only. 14. Hansen PA, Wang W, Marshall BA, Holloszy JO, Mueckler M: Dissociation
of GLUT4 translocation and insulin-stimulated glucose transport in trans-
ing ultimately to a significant reduction in insulin-mediated genic mice overexpressing GLUT1 in skeletal muscle. J Biol Chem
GLUT4 translocation. We hypothesize that basal glucose 273:18173–18179, 1998
uptake is elevated as an adaptive response, probably as a 15. Kahn BB, Simpson IA, Cushman SW: Divergent mechanisms for the insulin
resistant and hyperresponsive glucose transport in adipose cells from
result of elevated p38 MAPK phosphorylation and activity. fasted and refed rats: alterations in both glucose transporter number and
However, an additional defect was introduced into the p38 intrinsic activity. J Clin Invest 82:691– 699, 1988
MAPK signal pathway by the high Glc/Ins pretreatment, 16. Cuenda A, Rouse J, Doza YN, Meier R, Cohen P, Gallagher TF, Young PR,
resulting in a decrease in acute insulin-mediated gain in Lee JC: SB 203580 is a specific inhibitor of a MAP kinase homologue which
p38 MAPK phosphorylation and activity. These results is stimulated by cellular stresses and interleukin-1. FEBS Lett 364:229 –233,
1995
support the tenet that p38 MAPK may be a regulator of 17. Somwar R, Kim DY, Sweeney G, Huang C, Niu W, Lador C, Ramlal T, Klip
glucose uptake and is altered in states of insulin resis- A: GLUT4 translocation precedes the stimulation of glucose uptake by
tance. insulin in muscle cells: potential activation of GLUT4 via p38 mitogen-
activated protein kinase. Biochem J 359:639 – 649, 2001
18. Sweeney G, Somwar R, Ramlal T, Volchuk A, Ueyama A, Klip A: An
ACKNOWLEDGMENTS
inhibitor of p38 mitogen-activated protein kinase prevents insulin-stimu-
This work was supported by a research grant from the lated glucose transport but not glucose transporter translocation in 3T3–L1
Canadian Diabetes Association to A.K. C.H. was supported adipocytes and L6 myotubes. J Biol Chem 274:10071–10078, 1999
by a Canadian Pediatric Endocrinology Fellowship. R.S. 19. Sargeant RJ, Paquet MR: Effect of insulin on the rates of synthesis and
degradation of GLUT1 and GLUT4 glucose transporters in 3T3–L1 adipo-
was supported by a Doctoral Award from the Canadian cytes. Biochem J 290:913–919, 1993
Institute for Health Research. N.P. was supported by a 20. Van Bueren AM, Moholt-Siebert M, Begley DE, McCall AL: An immuniza-
studentship from the Banting and Best Diabetes Center at tion method for generation of high affinity antisera against glucose
the University of Toronto. W.N. was supported by a transporters useful in immunohistochemistry. Biochem Biophys Res Com-
mun 197:1492–1498, 1993
fellowship from the Visiting Scientist Program at the 21. Ueyama A, Yaworsky KL, Wang Q, Ebina Y, Klip A: GLUT-4myc ectopic
Hospital for Sick Children. expression in L6 myoblasts generates a GLUT-4-specific pool conferring
We thank Dr. Phil Bilan for helpful discussions and insulin sensitivity. Am J Physiol 277:E572–E578, 1999
careful review of the manuscript and Dr. Assaf Rudich for 22. Li D, Randhawa VK, Patel N, Hayashi M, Klip A: Hyperosmolarity reduces
thoughtful suggestions. glut4 endocytosis and increases its exocytosis from a vamp2-independent
pool in L6 muscle cells. J Biol Chem 276:22883–22891, 2001
23. Wang Q, Khayat Z, Kishi K, Ebina Y, Klip A: GLUT4 translocation by insulin
REFERENCES in intact muscle cells: detection by a fast and quantitative assay. FEBS Lett
1. Shulman GI: Cellular mechanisms of insulin resistance. J Clin Invest 427:193–197, 1998
106:171–176, 2000 24. Somwar R, Sumitani S, Taha C, Sweeney G, Klip A: Temporal activation of
2. Zierath JR, Krook A, Wallberg-Henriksson H: Insulin action and insulin p70 S6 kinase and Akt1 by insulin: PI 3-kinase-dependent and -independent
resistance in human skeletal muscle. Diabetologia 43:821– 835, 2000 mechanisms. Am J Physiol 275:E618 –E625, 1998
3. Garvey WT, Olefsky JM, Matthaei S, Marshall S: Glucose and insulin 25. Yaworsky K, Somwar R, Ramlal T, Tritschler HJ, Klip A: Engagement of the
co-regulate the glucose transport system in primary cultured adipocytes: a insulin-sensitive pathway in the stimulation of glucose transport by
new mechanism of insulin resistance. J Biol Chem 262:189 –197, 1987 alpha-lipoic acid in 3T3–L1 adipocytes. Diabetologia 43:294 –303, 2000
4. Kozka IJ, Clark AE, Holman GD: Chronic treatment with insulin selectively 26. Robinson R, Robinson LJ, James DE, Lawrence JC Jr: Glucose transport in
down-regulates cell-surface GLUT4 glucose transporters in 3T3–L1 adipo- L6 myoblasts overexpressing GLUT1 and GLUT4. J Biol Chem 268:22119 –
cytes. J Biol Chem 266:11726 –11731, 1991 22126, 1993

DIABETES, VOL. 51, JULY 2002 2097

 INSULIN RESISTANCE AND GLUT4 ACTIVITY

27. Mitsumoto Y, Klip A: Development regulation of the subcellular distribu- skeletal muscle from NIDDM subjects after in vivo insulin stimulation.
tion and glycosylation of GLUT1 and GLUT4 glucose transporters during Diabetes 46:524 –527, 1997
myogenesis of L6 muscle cells. J Biol Chem 267:4957– 4962, 1992 39. Goodyear LJ, Giorgino F, Sherman LA, Carey J, Smith RJ, Dohm GL:
28. Wilson CM, Mitsumoto Y, Maher F, Klip A: Regulation of cell surface Insulin receptor phosphorylation, insulin receptor substrate-1 phosphory-
GLUT1, GLUT3, and GLUT4 by insulin and IGF-I in L6 myotubes. FEBS lation, and phosphatidylinositol 3-kinase activity are decreased in intact
Lett 368:19 –22, 1995 skeletal muscle strips from obese subjects. J Clin Invest 95:2195–2204,
29. Murata H, Hruz PW, Mueckler M: The mechanism of insulin resistance 1995
caused by HIV protease inhibitor therapy. J Biol Chem 275:20251–20254, 40. Clark SF, Molero JC, James DE: Release of insulin receptor substrate
2000 proteins from an intracellular complex coincides with the development of
30. Nolte LA, Yarasheski KE, Kawanaka K, Fisher J, Le N, Holloszy JO: The insulin resistance. J Biol Chem 275:3819 –3826, 2000
HIV protease inhibitor indinavir decreases insulin- and contraction-stimu- 41. Zierler K: Does insulin-induced increase in the amount of plasma mem-
lated glucose transport in skeletal muscle. Diabetes 50:1397–1401, 2001 brane GLUTs quantitatively account for insulin-induced increase in glu-
31. Raingeaud J, Gupta S, Rogers JS, Dickens M, Han J, Ulevitch RJ, Davis RJ: cose uptake? Diabetologia 41:724 –730, 1998
Pro-inflammatory cytokines and environmental stress cause p38 mitogen- 42. Clark AE, Holman GD, Kozka IJ: Determination of the rates of appearance
activated protein kinase activation by dual phosphorylation on tyrosine and loss of glucose transporters at the cell surface of rat adipose cells.
and threonine. J Biol Chem 270:7420 –7426, 1995 Biochem J 278: 235–241, 1991
32. Saltiel AR: New perspectives into the molecular pathogenesis and treat- 43. Somwar R, Niu W, Kim DY, Sweeney G, Randhawa VK, Huang C, Ramlal T,
ment of type 2 diabetes. Cell 104:517–529, 2001 Klip A: Differential effects of phosphatidylinositol 3-kinase inhibition on
33. Maier VH, Gould GW: Long-term insulin treatment of 3T3–L1 adipocytes intracellular signals regulating GLUT4 translocation and glucose transport.
results in mis-targeting of GLUT4: implications for insulin-stimulated J Biol Chem 276:46079 – 46087, 2001
glucose transport. Diabetologia 43:1273–1281, 2000 44. Kayali AG, Austin DA, Webster NJ: Stimulation of MAPK cascades by
34. Nolan JJ, Freidenberg G, Henry R, Reichart D, Olefsky JM: Role of human insulin and osmotic shock: lack of an involvement of p38 mitogen-
skeletal muscle insulin receptor kinase in the in vivo insulin resistance of activated protein kinase in glucose transport in 3T3–L1 adipocytes. Dia-
noninsulin-dependent diabetes mellitus and obesity. J Clin Endocrinol betes 49:1783–1793, 2000
Metab 78:471– 477, 1994 45. Chen D, Elmendorf JS, Olson AL, Li X, Earp HS, Pessin JE: Osmotic shock
35. Maegawa H, Shigeta Y, Egawa K, Kobayashi M: Impaired autophosphory- stimulates GLUT4 translocation in 3T3L1 adipocytes by a novel tyrosine
lation of insulin receptors from abdominal skeletal muscles in nonobese kinase pathway. J Biol Chem 272:27401–27410, 1997
subjects with NIDDM. Diabetes 40:815– 819, 1991 46. Somwar R, Perreault M, Kapur S, Taha C, Sweeney G, Ramlal T, Kim DY,
36. Carvalho E, Rondinone C, Smith U: Insulin resistance in fat cells from Keen J, Cote CH, Klip A, Marette A: Activation of p38 mitogen-activated
obese Zucker rats: evidence for an impaired activation and translocation of protein kinase alpha and beta by insulin and contraction in rat skeletal
protein kinase B and glucose transporter 4. Mol Cell Biochem 206:7–16, muscle: potential role in the stimulation of glucose transport. Diabetes
2000 49:1794 –1800, 2000
37. Carvalho E, Jansson PA, Nagaev I, Wenthzel AM, Smith U: Insulin 47. Moxham CM, Tabrizchi A, Davis RJ, Malbon CC: Jun N-terminal kinase
resistance with low cellular IRS-1 expression is also associated with low mediates activation of skeletal muscle glycogen synthase by insulin in
GLUT4 expression and impaired insulin-stimulated glucose transport. vivo. J Biol Chem 271:30765–30773, 1996
FASEB J 15:1101–1103, 2001 48. Begum N, Ragolia L: High glucose and insulin inhibit VSMC MKP-1
38. Bjornholm M, Kawano Y, Lehtihet M, Zierath JR: Insulin receptor sub- expression by blocking iNOS via p38 MAPK activation. Am J Physiol
strate-1 phosphorylation and phosphatidylinositol 3-kinase activity in 278:C81–C91, 2000

2098 DIABETES, VOL. 51, JULY 2002