Insulin induced glut 4 translocation in high fat fed rats 2001

Defective Insulin-Induced GLUT4 Translocation in
Skeletal Muscle of High Fat–Fed Rats Is Associated
With Alterations in Both Akt/Protein Kinase B and
Atypical Protein Kinase C (␨/␭) Activities
Fre´de´ric Tremblay,1,2
Charles Lavigne,2,3
He´le`ne Jacques,2,3
and Andre´ Marette1,2
The cellular mechanism by which high-fat feeding in-
duces skeletal muscle insulin resistance was investi-
gated in the present study. Insulin-stimulated glucose
transport was impaired (ϳ40–60%) in muscles of high
fat–fed rats. Muscle GLUT4 expression was significantly
lower in these animals (ϳ40%, P < 0.05) but only in
type IIa–enriched muscle. Insulin stimulated the trans-
location of GLUT4 to both the plasma membrane and the
transverse (T)-tubules in chow-fed rats. In marked
contrast, GLUT4 translocation was completely abro-
gated in the muscle of insulin-stimulated high fat–fed
rats. High-fat feeding markedly decreased insulin recep-
tor substrate (IRS)-1–associated phosphatidylinositol
(PI) 3-kinase activity but not insulin-induced tyrosine
phosphorylation of the insulin receptor and IRS pro-
teins in muscle. Impairment of PI 3-kinase function was
associated with defective Akt/protein kinase B kinase
activity (؊40%, P < 0.01) in insulin-stimulated muscle
of high fat–fed rats, despite unaltered phosphorylation
(Ser473/Thr308) of the enzyme. Interestingly, basal
activity of atypical protein kinase C (aPKC) was ele-
vated in muscle of high fat–fed rats compared with
chow-fed controls. Whereas insulin induced a twofold
increase in aPKC kinase activity in the muscle of chow-
fed rats, the hormone failed to further increase the
kinase activity in high fat–fed rat muscle. In conclusion,
it was found that GLUT4 translocation to both the
plasma membrane and the T-tubules is impaired in the
muscle of high fat–fed rats. We identified PI 3-kinase as
the first step of the insulin signaling pathway to be
impaired by high-fat feeding, and this was associated
with alterations in both Akt and aPKC kinase activities.
Diabetes 50:1901–1910, 2001
I
nsulin resistance represents a major pathogenic
impairment in the development of type 2 diabetes
(1,2). In humans and rodents, skeletal muscle is the
primary site of insulin-mediated glucose disposal
(2). Insulin increases glucose uptake in muscle by eliciting
GLUT4 translocation from an intracellular storage site to
both the plasma membrane and the transverse (T)-tubules
through a complex signaling cascade (3–5). Impaired GLUT4
translocation has been shown to be linked to reduced
glucose utilization in muscle of insulin-resistant and type 2
diabetic subjects (6–8). However, the precise mechanism
underlying the reduced stimulatory effect of insulin on
glucose transport is still unclear. Both receptor and pos-
treceptor defects have been observed in various models of
insulin resistance (9).
Insulin stimulates GLUT4 translocation by binding its
receptor ␣-subunits, leading to autophosphorylation of the
transmembrane ␤-subunits and intrinsic activation of re-
ceptor tyrosine kinase activity. In skeletal muscle, the
activated insulin receptor (IR) increases the tyrosine phos-
phorylation of IR substrate (IRS)-1 and IRS-2, leading to
activation of phosphatidylinositol (PI) 3-kinase (4). It is
believed that PI 3-kinase activation by insulin is essential
for the stimulation of GLUT4 translocation. Indeed, a large
number of studies have shown that inhibitors of PI 3-ki-
nase (wortmannin and LY294002) or overexpression of a
mutated p85 adapter subunit lacking the ability to bind the
p110 catalytic subunit fully inhibit insulin-mediated GLUT4
translocation and glucose transport in adipocytes and
skeletal muscle cells (rev. in [10]).
Downstream effector(s) of PI 3-kinase involved in the
regulation of glucose transport have yet to be clearly
identified. Candidate molecules of interest include the
serine/threonine kinase Akt (also termed protein kinase B
[PKB] or related to A and C [RAC] protein kinase) and
atypical protein kinase C (aPKC) (␨/␭). Both aPKC and Akt
lie in the PI 3-kinase/3-phosphoinositide-dependent kinase
(PDK)-1 signaling pathway, giving rise to phosphorylation
on Thr410 and Thr308, respectively (11,12). Full activation
of Akt further requires phosphorylation on Ser473 by the
putative PDK-2 (13), whereas aPKC activity appeared to be
dependent on autophosphorylation by an as yet unknown
mechanism (14). Evidence for the implication of Akt and
aPKC in the insulin-dependent regulation of glucose trans-
From the 1
Department of Physiology and 2
Lipid Research Unit, Laval Univer-
sity Hospital Research Center; and the 3
Department of Food Science and
Nutrition, Human Nutrition Research Group, Laval University, Ste-Foy, Que´-
bec, Canada.
Address correspondence and reprint requests to Andre´ Marette, Depart-
ment of Physiology and Lipid Research Unit, Laval University Hospital
Research Center, 2705, Laurier Blvd., Ste-Foy, Que´bec, Canada, G1V 4G2.
Email: andre.marette@crchul.ulaval.ca.
Received for publication 30 June 2000 and accepted in revised form 7 May
2001.
aPKC, atypical protein kinase C; DTT, dithiothreitol; 2-[3
H]DG, 2-deoxy-
D-[3
H]glucose; IR, insulin receptor; IRS, insulin receptor substrate; MAP,
mitogen-activated protein; PBS, phosphate-buffered saline; PDK, 3-phospho-
inositide-dependent kinase; PI, phosphatidylinositol; PKB, protein kinase B;
PKC, protein kinase C; PVDF, polyvinylidene difluoride; RAC, related to A and
C; RDU, relative densitometric units; T, transverse; TNF-␣, tumor necrosis
factor-␣.
DIABETES, VOL. 50, AUGUST 2001 1901

port in muscle cells arises from transfection studies using
either kinase-inactive or overexpression/constitutively ac-
tive forms of the kinases (15–19). Only a few studies have
examined insulin-dependent activation of Akt in muscle
from animal models of insulin resistance and diabetic
subjects, and these yielded contradictory results regarding
its possible involvement in impaired glucose homeostasis
(20–23). However, it is still unknown whether the stimu-
latory effect of insulin on aPKC is altered in muscles from
insulin-resistant animals or humans.
The high fat–feeding model of insulin resistance dis-
plays common features of the abdominal obesity syn-
drome encountered in insulin-resistant subjects (24,25).
Indeed, rats fed a high-fat diet develop skeletal muscle
insulin resistance, increased adiposity, hyperinsulinemia,
and mild hyperglycemia (24–27). It is generally believed
that GLUT4 protein expression is normal in skeletal mus-
cle of high fat–fed rats (24,27,28), but this has not been a
consistent finding (29–31). Recruitment of GLUT4 assessed
by exofacial photolabelling (2-N-4-(1-azi2,2,2-trifluoro-
ethyl)-benzoyl-1,3-bis-(D-mannose-4-yloxy)-2-propylamine)
led to the view that a high-fat diet reduced insulin-
stimulated GLUT4 translocation to the cell surface (27,32).
However, Rosholt et al. (28) did not observe such impair-
ment in isolated plasma membrane vesicles, suggesting
that the main site of defective GLUT4 translocation may be
localized to the T-tubules, the principal component of the
muscle cell surface, although this remains to be studied.
Early impairments in the insulin signaling cascade may
lead to a decrease in GLUT4 translocation in muscle of
high fat–fed rats. Insulin-induced tyrosine phosphorylation
of both the IR and IRS-1 was reported to be normal in
rats fed a high-fat diet for 8 weeks (32). However, IRS-1
associated PI 3-kinase activity has been found to be im-
paired in mice after 4 weeks of high-fat feeding (27).
Whether insulin-dependent activation of either Akt or aPKC
pathways is altered in skeletal muscle of high fat–fed rats
is still unknown.
The aim of the present study was to clarify the cellular
mechanisms leading to defective insulin-stimulated glu-
cose transport in skeletal muscle of the high fat–fed rat.
More specifically, we tested the hypothesis that poten-
tial alterations of signaling elements downstream of PI
3-kinase activation may be linked with impaired GLUT4
translocation in the muscle of high fat–fed obese rats.
RESEARCH DESIGN AND METHODS
Materials. Reagents for SDS-PAGE and immunoblotting were from Bio-Rad
(Mississauga, ON, Canada). Enhanced chemiluminescence reagent, 2-deoxy-
D-[3
H]glucose (2-[3
H]DG), and D-14
C-sucrose were from NEN Life Science
Products (Boston, MA). [␥-32
P]ATP, protein A- and G-Sepharose, and anti-
mouse or anti-rabbit IgG conjugated to horseradish-peroxidase were pur-
chased from Amersham Pharmacia Biotech (Baie d’Urfe´, QC, Canada).
Anti-goat IgG conjugated to horseradish-peroxidase, polyclonal antibodies
against GLUT1 (raised against 20 COOH-terminal amino acids [C-20]), GLUT4
(C-20), IRS-1 (C-20), aPKCs (C-20), and Akt 1/2 (which recognizes both Akt 1
and 2 [H-136]), were obtained from Santa Cruz Biotechnology (Santa Cruz,
CA). Anti–phospho-specific (Ser473 and Thr308) antibodies against Akt were
from New England Biolabs (Beverly, MA). Akt/PKB substrate (Crosstide) and
antibodies against phosphotyrosine (4G10 clone) and p85 were obtained from
Upstate Biotechnology (Lake Placid, NY). Myelin basic protein was from
Sigma (St. Louis, MO). Okadaic acid was purchased from Calbiochem (La
Jolla, CA). L-␣–PI was from Avanti Polar Lipids (Alabaster, AL). Oxalate-
treated thin-layer chromatography silica gel H plates were obtained from
Analtech (Newark, DE). All other chemicals were of the highest analytical
grade.
Treatment of animals. All experiments reported here were approved by the
Laval University Animal Care and Handling Committee and comply with
Canadian Council on Animal Care guidelines for the care and use of animals
for research purposes. Male Wistar rats (Charles River, Montreál, QC, Canada)
weighing 200–250 g at the beginning of the study were housed individually in
plastic cages in animal quarters maintained at 22°C with a 12:12-h dark-light
schedule. Animals were fed either low-fat Rodent Chow (Charles River Ro-
dent Chow 5075, Purina Mills, St. Louis, MO) or a purified high-fat diet for
4 weeks. As percent of total energy, the high-fat diet consisted of 32.5% lard,
32.5% corn oil, 20% sucrose, and 15% protein, whereas the Rodent Chow diet
contained 57.3% carbohydrate, 18.1% protein, and 4.5% fat. The energy con-
tents of the diets were 14.3 kJ/g for the Rodent Chow diet and 25.5 kJ/g for the
high-fat diet.
Hyperinsulinemic-euglycemic clamp and tracer injection. The clamp
procedure was essentially performed as previously described (33). Briefly,
unrestrained conscious animals were allowed to rest for 40 min before the
initial blood sample (300 ␮l) was obtained. For hyperinsulinemic-euglycemic
clamp, a continuous intravenous infusion of insulin was then started at the
rate of 4 mU ⅐ kgϪ1
⅐ minϪ1
and continued for 2 h. The arterial blood glucose
concentration was clamped using a variable-rate glucose infusion. Control
rats were infused with saline for the same period of time, and no exogenous
glucose was necessary to maintain euglycemia. Tracer injection (2-[3
H]DG and
14
C-sucrose) was administered 20 min before the end of the clamp to deter-
mine individual tissue glucose uptake, as previously reported (33). Immedi-
ately after the clamp, the rats were killed, and their hindlimb muscles (soleus,
tibialis, gastrocnemius, and quadriceps) were rapidly excised, cleaned of
extraneous tissues, and frozen in liquid nitrogen. The muscle were kept at
Ϫ80°C until further processing.
Acute insulin stimulation. Overnight-fasted rats were injected either with
saline or insulin (8 units/kg) for 4 min, as previously described (3). Muscles
were quickly excised and immediately frozen in liquid nitrogen. Muscles were
homogenized in six volumes of lysis buffer containing 20 mmol/l Tris, pH 7.5,
140 mmol/l NaCl, 1 mmol/l CaCl2, 1 mmol/l MgCl2, 10% glycerol, 10 mmol/l
sodium pyrophosphate, 10 mmol/l NaF, 2 mmol/l Na3VO4, 2 mg/ml benzami-
dine, 1 mmol/l PMSF, and protease inhibitor cocktail (Sigma). Okadaic acid
(100 nmol/l) was added in lysis buffer for Akt and aPKC kinase activities.
Muscle homogenates were solubilized in 1% Nonidet P-40 for 1 h at 4°C
and centrifuged at 14,000g for 10 min. Supernatant was used for insulin
signaling studies as described below.
Subcellular fractionation. Plasma membranes, T-tubules, and GLUT4-
enriched intracellular membranes were isolated from muscles (8–10 g, mixed
gactrocnemius and quadriceps) using a procedure developed in our laboratory
(3,34). This subcellular fractionation protocol has been extensively character-
ized with immunologic and enzymatic markers (3,34). In brief, this technique
allows the simultaneous and separated isolation of plasma membrane, T-
tubules, and intracellular membrane vesicles from the same muscle homoge-
nate. GLUT4 content was determined in fractions obtained from saline- or
insulin-infused rats by Western blotting, as described below.
Western blotting. Membranes (10 ␮g) or muscle homogenates (50 ␮g) were
subjected to SDS-PAGE (7.5% gel) and electrophoretically transferred to
polyvinylidene difluoride (PVDF) filter membranes for 2 h. PVDF membranes
were then blocked for 1 h at room temperature with buffer I (50 mmol/l
Tris-HCl, pH 7.4, and 150 mmol/l NaCl) containing 0.04% NP-40, 0.02%
Tween-20, and 5% nonfat milk. This step was followed by overnight incubation
at 4°C with primary antibodies, as described in the figure legends. The PVDF
membranes were then washed for 30 min, followed by a 1-h incubation with
either anti-mouse or anti-rabbit IgG conjugated to horseradish-peroxidase in
buffer I containing 1% bovine serum albumin. The PVDF membranes were
washed for 30 min in buffer I, and the immunoreactive bands were detected by
the enhanced chemiluminescence method. A muscle standard (an unrelated
crude membrane fraction) was run on every gel for comparison of samples
from different immunoblots.
Tyrosine phosphorylation of the IR and IRS. Muscle lysates (1 mg of
protein) were immunoprecipitated with 2 ␮g of anti-phosphotyrosine (4G10)
coupled to protein A-Sepharose overnight at 4°C. The immune complex was
washed three times in phosphate-buffered saline (PBS) (pH 7.4) containing 1%
NP-40 and 2 mmol/l Na3VO4, resuspended in Laemmli buffer, and boiled for
5 min. Proteins were resolved on SDS-PAGE (6% gel) and processed for
Western blot analysis as described above.
PI 3-kinase activity. Muscle lysates (1 mg of protein) were immunoprecipi-
tated with 2 ␮g of anti–IRS-1 coupled to protein A-Sepharose overnight at 4°C.
PI 3-kinase activity was determined as described by Kristiansen et al. (35),
with minor modifications. Immune complexes were washed twice with wash
buffer I (PBS, pH 7.4, 1% NP-40, and 2 mmol/l Na3VO4), twice with wash buffer
II (100 mmol/l Tris, pH 7.5, 500 mmol/l LiCl, and 2 mmol/l Na3VO4), and twice
with wash buffer III (10 mmol/l Tris, pH 7.5, 100 mmol/l NaCl, 1 mmol/l EDTA,
INSULIN SIGNALING IN MUSCLE OF HIGH FAT–FED RATS
1902 DIABETES, VOL. 50, AUGUST 2001

and 2 mmol/l Na3VO4). Beads were resuspended in 70 ␮l of kinase buffer (8
mmol/l Tris, pH 7.5, 80 mmol/l NaCl, 0.8 mmol/l EDTA, 15 mmol/l MgCl2, 180
␮mol/l ATP, and 5 ␮Ci [␥-32
P]ATP) and 10 ␮l of sonicated PI mixture (20 ␮g
L-␣-PI, 10 mmol/l Tris, pH 7.5, and 1 mmol/l EGTA) for 15 min at 30°C.
Reaction was stopped by the addition of 20 ␮l 8 mol/l HCl, mixed with 160 ␮l
CHCl3:CH3OH (1:1), and centrifuged. Lower organic phase was spotted on
oxalate-treated silica gel TLC plates and developed in CHCl3:CH3OH:H2O:
NH4OH (60:47:11.6:2). The plate was dried and visualized by autoradiography
with intensifying screen at Ϫ80°C.
Akt/PKB activity. Muscle lysates (1 mg of protein) were immunoprecipitated
with 4 ␮g of anti–Akt 1/2 coupled to protein G-Sepharose for 4 h at 4°C. Akt
activity was measured essentially as described previously (20,21). Immune
complexes were washed twice with wash buffer I (PBS, pH 7.4, 1% NP-40, and
100 ␮mol/l Na3VO4) and twice with wash buffer II (50 mmol/l Tris, pH 7.5, 10
mmol/l MgCl2, and 1 mmol/l dithiothreitol [DTT]). Beads were resuspended in
30 ␮l of kinase buffer (50 mmol/l Tris, pH 7.5, 10 mmol/l MgCl2, 1 mmol/l DTT,
8 ␮mol/l ATP, 2 ␮Ci [␥-32
P]ATP, and 50 ␮mol/l Crosstide) for 30 min at 30°C.
Reaction product was resolved on a 40% acrylamide-urea gel and visualized by
autoradiography with intensifying screen at Ϫ80°C. In preliminary experi-
ments in which insulin was injected in intact rats for 4, 10, 20, or 30 min, it was
determined that Akt/PKB kinase activation by insulin was maximal after 4 min
of insulin injection (data not shown).
aPKC (␨/␭) activity. Muscle lysates (1 mg of protein) were immunoprecipi-
tated with 2 ␮g of anti-PKC (␨/␭) overnight at 4°C, then immune complexes
were collected on protein A/G-Sepharose for 2 h. aPKC activity was deter-
mined according to the method of Chou et al. (12). Beads were washed twice
with wash buffer I (PBS, pH 7.4, 1% NP-40, and 2 mmol/l Na3VO4), twice with
wash buffer II (100 mmol/l Tris, pH 7.5, 500 mmol/l LiCl, and 100 ␮mol/l
Na3VO4), and twice with wash buffer III (50 mmol/l Tris, pH 7.5, 10 mmol/l
MgCl2, and 100 ␮mol/l Na3VO4). Beads were resuspended in 30 ␮l of kinase
buffer (50 mmol/l Tris, pH 7.5, 10 mmol/l MgCl2, 40 ␮mol/l ATP, 5 ␮Ci
[␥-32
P]ATP, and 5 ␮g myelin basic protein) for 12 min at 30°C. Reaction was
stopped by the addition of Laemmli buffer and heated for 30 min at 37°C.
Reaction product was resolved on 13% SDS-PAGE. Gel was dried and
visualized by autoradiography with intensifying screen at Ϫ80°C. In prelimi-
nary experiments, it was determined that aPKC activation by insulin was
maximal after 4 min of insulin injection (data not shown). Furthermore, we
have verified that neither the M-kinase form of aPKC (36) nor Akt 1/2 (37) or
PI 3-kinase (38) were recovered in the aPKC immunoprecipitates of both
Rodent Chow– and high fat–fed rats (data not shown).
Data analysis. Autoradiographs were analyzed by laser scanning densitom-
etry using a tabletop Agfa scanner (Arcus II; Agfa-Gavaert, Morstel, Belgium)
and quantified with the National Institutes of Health Image program (acces-
sible online at rsb.info.nih.gov/nih-image/). All data are presented as means Ϯ
SE. The effects of diets and insulin were compared by a two-way analysis of
variance. Differences were considered to be statistically significant at P Ͻ
0.05.
RESULTS
Physiological parameters of high fat–fed rats. As
expected, feeding rats a high-fat diet for 4 weeks resulted
in increased adiposity, hyperinsulinemia, and moderate
hyperglycemia (Table 1). High-fat diet–mediated loss of
insulin sensitivity was evidenced by the decrease in glu-
cose infusion rate (ϳ30%) during the euglycemic-hyperin-
sulinemic clamp (Table 1). These results are in close
agreement with previous studies showing the diabetogenic
effect of a high-fat diet (24–27).
Glucose transport and GLUT4 expression in individ-
ual muscles. The effect of fat feeding on GLUT4 expres-
sion and glucose transport in muscles enriched in different
fiber types is presented in Fig. 1A and B. Total GLUT4
content was greater (ϳ2.5-fold) in type I- and IIa-enriched
muscles (soleus and red tibialis, respectively) compared
with type IIb-enriched muscles (white gastrocnemius) in
chow-fed rats. These results are consistent with our pre-
vious observations that GLUT4 expression is higher in
oxidative than glycolytic fibers (39). In rats fed a high-fat
diet, GLUT4 content in type I- and IIb-enriched muscle was
found to be similar to chow-fed rats. However, GLUT4
abundance was reduced in type IIa-enriched muscles
(ϳ40%) (Fig. 1A), and this observation was confirmed in
FIG. 1. Comparison between GLUT4 protein expression and insulin-
stimulated glucose uptake in muscles enriched in different fiber types.
A: GLUT4 protein expression was measured in muscle homogenates
from type I-, type IIa-, and type IIb- (soleus, red tibialis, and white
gastrocnemius, respectively) enriched muscles of chow- and high fat–
fed rats. Proteins (50 ␮g) were resolved on 7.5% SDS-PAGE and
immunoblotted with polyclonal anti-GLUT4, as described in RESEARCH
DESIGN AND METHODS. The means ؎ SE from 4–10 determinations for each
muscle are shown. B: In vivo 2-[3
H]DG uptake in muscle fibers was
determined after bolus injection of 2-[3
H]DG and 14
C-sucrose in insu-
lin-infused rats (four mU ⅐ kg؊1
⅐ min؊1
) at the end of the euglycemic-
hyperinsulinemic clamp as described under RESEARCH DESIGN AND METHODS.
The means ؎ SE from 7–9 determinations are shown. Ⅺ, chow-fed; f,
high fat–fed. #P < 0.05 vs. chow-fed rats.
TABLE 1
Physiological parameters of rats fed a Rodent Chow– or a high
fat–diet
Rodent Chow High-Fat
Body weight (g) 351 Ϯ 20 380 Ϯ 20
Epididymal fat pad (g) 1.92 Ϯ 0.10 3.33 Ϯ 0.30*
Retroperitoneal fat pad (g) 1.17 Ϯ 0.15 2.67 Ϯ 0.35*
Fasting glucose (mmol/l) 7.2 Ϯ 0.3 8.9 Ϯ 0.2*
Fasting insulin (nmol/l) 0.13 Ϯ 0.02 0.25 Ϯ 0.03*
GIR (mg ⅐ kg–1
⅐ min–1
) 16.9 Ϯ 2.0 12.2 Ϯ 1.1*
Values are means Ϯ SE. GIR, glucose infusion rate. *P Ͻ 0.05 vs.
chow value.
F. TREMBLAY AND ASSOCIATES

the muscles used for the GLUT4 translocation assay
(mixed gastrocnemius and quadriceps), which are en-
riched (ϳ50%) with type IIa ﬁbers (Fig. 2A). Despite the
latter ﬁnding, we observed similar reductions (ϳ40–60%)
in insulin-stimulated glucose transport in all skeletal mus-
cles tested in high fat–fed rats (Fig. 1B), whereas basal
glucose transport was unaltered by fat feeding (data not
shown). These results indicate that the reduced expres-
sion of GLUT4 is not the principal cause of impaired
insulin-stimulated glucose transport in skeletal muscle of
high fat–fed rats.
GLUT4 translocation to the plasma membrane and
the T-tubules. We next investigated the effect of high-fat
feeding on GLUT4 translocation to both cell surface
compartments of muscle cells (i.e., the plasma membrane
and the T-tubules) to precisely determine the locus of
insulin resistance in this animal model (Fig. 2B–D). The
characteristics of the subcellular membrane fractions are
presented in Table 2 and are in good agreement with
previous studies (3,34,40). Insulin stimulation induced
translocation of GLUT4 from the intracellular membranes
(Ϫ35%, from 330 Ϯ 58 to 215 Ϯ 28 relative densitometric
units [RDU], P Ͻ 0.05) to the plasma membrane (ϩ100%,
from 98 Ϯ 18 to 196 Ϯ 28 RDU, P Ͻ 0.05) and the T-tubules
(ϩ35%, from 53 Ϯ 7 to 71 Ϯ 5 RDU, P Ͻ 0.05) in control
rats fed a standard chow diet. In marked contrast, insulin
failed to induce GLUT4 translocation to either the plasma
membrane or the T-tubules in skeletal muscle of rats fed
the high-fat diet (Fig. 2B–D). These results clearly show
that the reduced insulin-stimulated glucose uptake in
muscle of high fat–fed rats is linked to a defective trans-
location of GLUT4 glucose transporters to both cell sur-
face compartments of skeletal muscle cells.
Tyrosine phosphorylation of IR and IRS proteins. To
investigate whether high-fat feeding causes insulin resis-
tance via the alteration of an early insulin signaling step,
we measured insulin-induced tyrosine phosphorylation of
the IR and IRS proteins in anti-phosphotyrosine immune
complexes. In rats fed either the control chow or the
high-fat diets, insulin stimulated the tyrosine phosphory-
lation of IR and IRS proteins (approximately six- and
threefold, respectively) (Fig. 3A and B). Thus, high-fat
FIG. 2. Effect of high fat–feeding on GLUT4 translocation in skeletal muscle. Rats were clamped with either saline (basal) or insulin (4 mU ⅐ kg؊1
⅐ min؊1
) for 2 h. Immediately after the clamp, muscles (mixed gastrocnemius and quadriceps) were quickly excised, cleaned of extraneous tissues,
and frozen in liquid nitrogen. GLUT4 content was assessed by Western blotting in total homogenate (A), plasma membrane (B), T-tubules (C),
and intracellular membranes (D) isolated from saline- or insulin-infused rats as described in RESEARCH DESIGN AND METHODS. The means ؎ SE from
4–5 individual membrane preparations are shown. Ⅺ, Basal; f, insulin. IM, intracellular membranes; PM, plasma membrane; TT, T-tubules. #P <
0.05 vs. chow-fed rats. *P < 0.05 vs. corresponding basal value.
TABLE 2
Characterization of membrane fractions from skeletal muscle
Fractions Diet Insulin
Protein
recoveries
(␮g/g muscle)
5Ј-nucleotidase
(nmol ⅐ mg–1
⅐
min–1
)
PM Rodent Chow Ϫ 30 Ϯ 5 557 Ϯ 57
Rodent Chow ϩ 30 Ϯ 5 546 Ϯ 74
High-Fat Ϫ 38 Ϯ 5 417 Ϯ 138
High-Fat ϩ 31 Ϯ 4 355 Ϯ 77
T-tubules Rodent Chow Ϫ 234 Ϯ 24 90 Ϯ 7
Rodent Chow ϩ 279 Ϯ 12 72 Ϯ 8
High-Fat Ϫ 258 Ϯ 10 59 Ϯ 14
High-Fat ϩ 265 Ϯ 24 52 Ϯ 14
Rodent Chow Ϫ 95 Ϯ 21 ND
IM Rodent Chow ϩ 99 Ϯ 27 ND
High-Fat Ϫ 136 Ϯ 19 ND
High-Fat ϩ 219 Ϯ 45 ND
Values are means Ϯ SE. PM, plasma membranes; IM, Intracellular
membranes; ND, nondetectable.

feeding for 4 weeks did not affect the stimulatory effect of
insulin on IR/IRS tyrosine phosphorylation in skeletal
muscle.
PI 3-kinase activity. Although high-fat feeding did not
alter the activation of proximal events in insulin signaling
(IR/IRS), we next evaluated insulin-induced activation
of PI 3-kinase, a lipid kinase that mediates most of the
metabolic action of insulin (10). Protein levels of PI
3-kinase p85 subunit were similar in muscle from chow-
and high fat–fed rats (0.92 Ϯ 0.04 and 0.81 Ϯ 0.05 RDU,
respectively; NS). The kinase activity of the enzyme was
measured in anti–IRS-1 precipitates because IRS-1 is the
main isoform responsible for insulin-stimulated glucose
transport in skeletal muscle (41,42). In the basal state, PI
3-kinase activity was not different among the dietary
groups (Fig. 4). Following insulin stimulation, PI 3-kinase
activity in the muscle of control rats was markedly in-
creased (approximately eightfold). However, in muscle of
rats fed the high-fat diet, insulin-dependent PI 3-kinase
activation was severely attenuated (ϳ60% reduction ver-
sus chow-fed rats, P Ͻ 0.05).
Akt phosphorylation and activity. Because impair-
ment of PI 3-kinase stimulation by insulin may lead to a
concomitant decrease in the activation of downstream
effector(s) in rats fed a high-fat diet, we next measured
insulin-induced phosphorylation of Akt using phospho-
specific (Ser473 and Thr308) antibodies. Akt phosphoryla-
tion was robustly enhanced by insulin in skeletal muscle of
rats fed the standard chow diet. The effect of insulin was
similar in high fat–fed rats (Fig. 5A). We then assessed the
kinase activity of the enzyme in anti-Akt immunoprecipi-
tates using Crosstide (a peptide containing a glycogen
synthase kinase-3 motif known to be phosphorylated by
Akt) as substrate (43). We observed that basal Akt kinase
activity tended to be reduced (ϳ50%, P ϭ 0.16) in high
fat–fed animals compared with control rats. Insulin in-
creased Akt kinase activity by ϳ2.2-fold in both dietary
groups compared with their respective basal activity (Fig.
5B). However, maximal activation of Akt (insulin-treated
groups) was reduced by ϳ40% (P Ͻ 0.01) in rats fed the
high-fat diet compared with chow-fed rats (Fig. 5B).
Furthermore, the reduced Akt kinase activity could not be
attributed to a decrease in Akt protein expression in the
muscle of high fat–fed rats (0.93 Ϯ 0.10 and 0.82 Ϯ 0.07
RDU for chow- and high fat–fed rats, respectively; NS).
aPKC activity and translocation. Another downstream
FIG. 3. Effect of high-fat feeding on insulin-induced tyrosine phosphor-
ylation of IR/IRS proteins. Overnight-fasted rats were injected with
either saline or insulin (8 units/kg) for 4 min. Phosphoproteins were
immunoprecipitated from muscle homogenates, resolved on 6% SDS-
PAGE, and immunoblotted using anti-phosphotyrosine (4G10) as de-
scribed in RESEARCH DESIGN AND METHODS. Quantification of tyrosine
phosphorylation of the IR (A) and IRS (B) proteins was expressed
relative to chow-fed basal values. Representative immunoblots are
shown at the top of each panel. The location of molecular weight
markers is shown on the right. The means ؎ SE from 4–6 determina-
tions from different animals are shown. Ⅺ, Basal; f, insulin.
FIG. 4. Effect of high-fat feeding on PI 3-kinase activity. PI 3-kinase
was measured in anti–IRS-1 immunoprecipitates of muscle homogenate
as described in RESEARCH DESIGN AND METHODS. Quantification of 32
P
incorporated into PI 3-phosphate was expressed relative to chow-fed
basal values. A representative autoradiograph is shown at the top of
the figure. The means ؎ SE of 4–6 determinations from different
animals are shown. Ⅺ, Basal; f, insulin. *P < 0.05 vs. corresponding
basal value; #P < 0.05 vs. chow-fed insulin values. PI(3)P, PI 3-phos-
phate.

effector of the PI 3-kinase pathway is the diacylglycerol-
and calcium-insensitive aPKC (␨/␭). We first examined if
insulin stimulated the kinase activity of aPKC in skeletal
muscle. We found that insulin stimulates aPKC activity in
anti-PKC (␨/␭) complexes by 2.2-fold (P Ͻ 0.01), as mea-
sured by 32
P incorporated in myelin basic protein (Fig.
6A). We next determined whether defective PI 3-kinase
activity in skeletal muscle of high fat–fed rats was linked
to impaired aPKC activation by insulin. In marked contrast
to the situation observed in chow-fed animals, aPKC
activity was already elevated in control muscles of high
fat–fed rats. Moreover, insulin could not further activate
the enzyme in the muscle of these insulin-resistant ani-
mals. aPKC has been shown to be translocated to mem-
branes when activated by insulin (14). Accordingly, we
also found that insulin increases the association of aPKC
with the plasma membrane (ϳ50%, P Ͻ 0.01) in the
skeletal muscle of chow-fed rats after a hyperinsulinemic
clamp (Fig. 6B). As observed for aPKC kinase activity,
high-fat feeding in rats was found to increase basal aPKC
association with the plasma membrane as compared with
chow-fed rats (ϩ35%, P Ͻ 0.05), and insulin infusion failed
to further increase this association in high fat–fed rats.
Muscle expression of aPKC protein was similar between
both groups (1.11 Ϯ 0.36 and 1.01 Ϯ 0.15 RDU for chow-
and high fat–fed rats, respectively; NS).
DISCUSSION
The cellular mechanism(s) responsible for impaired insu-
lin-stimulated glucose uptake in the peripheral tissues of
insulin-resistant subjects is still unclear. In the present
study, we used the high fat–fed rat model to clarify the
cellular defects behind the occurrence of skeletal muscle
FIG. 5. Effect of high-fat feeding on Akt/PKB phosphorylation and
activity. Overnight fasted rats were injected with either saline or
insulin (8 units/kg) for 4 min. A: Phosphorylation state of Akt (Ser473
and Thr308) was measured in muscle homogenates. Protein (50 ␮g)
was separated on 7.5% SDS-PAGE and immunoblotted with anti–
phospho-specific antibody against Akt as described in RESEARCH DESIGN
AND METHODS. B: Akt kinase activity was measured in anti–Akt-1/2
immunoprecipitates as described in RESEARCH DESIGN AND METHODS. Quan-
tification of 32
P incorporated into Crosstide was expressed relative to
chow-fed basal values. Representative immunoblot (A) and autoradio-
graph (B) are shown at the top of each panel. The means ؎ SE of 4–6
determinations from different animals are shown. Ⅺ, Basal; f, insulin.
*P < 0.05 vs. corresponding basal value; #P < 0.01 vs. chow-fed insulin
values.
FIG. 6. Effect of high-fat feeding on aPKC activity and translocation.
Overnight-fasted rats were injected either with saline or insulin (8
units/kg) for 4 min. A: aPKC kinase activity was measured in anti-PKC
(␨␭) immunoprecipitates as described in RESEARCH DESIGN AND METHODS.
Quantification of 32
P incorporated into myelin basic protein was ex-
pressed relative to chow-fed basal values. B: Membrane recovery of
aPKC in control and insulin-infused rats was assessed by Western blot-
ting as described in RESEARCH DESIGN AND METHODS. Representative
autoradiograph (A) and immunoblot (B) are shown at the top of each
panel. The location of molecular weight markers is shown on the right.
The means ؎ SE of 4–6 determinations from different animals are
shown. Ⅺ, Basal; f, insulin. *P < 0.01 vs. corresponding basal value;
#P < 0.05 vs. chow-fed basal values.

insulin resistance. We first looked at possible alterations in
GLUT4 expression and/or translocation because both are
important determinants of glucose uptake in adipocytes
and muscle cells (1). Whereas impaired glucose transport
in response to insulin in the adipose tissue of high fat–fed
rats has been attributed to decreased GLUT4 content
(44,45), discrepancies still exist regarding whether fat
feeding alters GLUT4 expression in skeletal muscle
(24,27–31). These discrepant findings may be partly ex-
plained by the fact that different types of muscles were
used in these studies.
In this study, we found that feeding a high-fat diet
caused a selective downregulation of GLUT4 in type IIa-
enriched muscle. Despite this, total GLUT4 content does
not appear to predict the extent of insulin resistance in the
muscle of high fat–fed rats. As shown in this study and in
the results from other studies (27,32), the cellular localiza-
tion of GLUT4, rather than its amount, is the principal
determinant of impaired insulin-stimulated glucose trans-
port in the muscle fibers of high fat–fed rats. We previously
observed that GLUT4 translocation was selectively im-
paired to the T-tubules but normal to the plasma mem-
brane of muscle from insulin-resistant type 1 diabetic rats
(40). In this study, we found that impaired GLUT4 trans-
location in rats fed a high-fat diet was generalized to both
cell surface compartments. There are currently no data
available concerning insulin-induced GLUT4 translocation
to the T-tubules in insulin-resistant subjects. Regulation of
GLUT4 translocation to this surface compartment is par-
ticularly important because the T-tubules cover most of
the cell surface area in muscle cells. Surprisingly, the
extent of insulin resistance on GLUT4 translocation (not
detectable) in high fat–fed animals was more pronounced
than that observed for glucose transport (40–60% inhibi-
tion). This discrepancy cannot be explained by a greater
contribution from the GLUT1 transporter in the muscle of
high fat–fed rats because its levels were also found to be
reduced (by 49%, P Ͻ 0.05) in the plasma membrane of
high fat–fed animals (GLUT1 was not detectable in the
T-tubules). Recently, Ryder et al. (8) reported similar
results in the muscle of type 2 diabetic subjects, showing
that the inhibition of GLUT4 translocation (ϳ90%) did not
match the reduction of insulin-stimulated glucose trans-
port (ϳ50%). These authors suggested that another insu-
lin-sensitive glucose transporter (e.g., the newly described
GLUTX1 [46]) may have partly rescued the lack of GLUT4
translocation to the cell surface. Alternatively, one might
speculate that in high fat–fed rats, insulin is still able to
promote glucose transport by increasing the intrinsic
activity of cell surface GLUT4 via the p38 mitogen-acti-
vated protein (MAP) kinase pathway (47). Indeed, it has
been recently reported that SB 203580, an inhibitor of the
p38 MAP kinase pathway, decreased glucose transport
activity in L6 myotubes and in rat skeletal muscle (47,48)
without interfering with GLUT4 translocation and inser-
tion at the cell surface, implying that it inhibited GLUT4
activation (47).
There are currently two models that have been pro-
posed to explain the defective insulin-induced activation
of glucose transport in the high fat–feeding model of
insulin resistance. On one hand, it has been reported that
an alteration of insulin signaling (i.e., activation of PI
3-kinase) was an early occurrence in the pathogenesis of
impaired glucose transport in the skeletal muscle of mice
fed a high-fat diet (27). On the other hand, it has been
suggested that defects in insulin signaling is a late event
and is not the primary defect causing muscle insulin
resistance in high fat–fed animals (32). The latter model
was based on the observation that IR function and IRS-1
phosphorylation were not affected in rats fed a high-fat
diet for 8 weeks, despite significant reductions in insulin-
mediated GLUT4 translocation. However, PI 3-kinase ac-
tivity was not assessed in the latter study.
In the present study, we found that impaired insulin-
stimulated glucose transport and GLUT4 translocation in
skeletal muscle of high fat–fed rats was associated with
defective PI 3-kinase activation. Furthermore, we con-
firmed that this defect occurred without alteration of
IR/IRS-1 tyrosine phosphorylation in the high-fat feeding
model of insulin resistance (32). The latter finding is
consistent with the fact that impairments of IR/IRS-1
signaling occurred later and are not the primary cause of
decreased insulin-stimulated glucose transport in this
model. A putative mechanism for the impairment of PI
3-kinase is an increased serine phosphorylation of IRS-1,
which in turn would act as a inhibitor of PI 3-kinase
function. For instance, 14–3-3 protein has been shown to
bind to phosphoserine residues of IRS-1 and subsequently
inhibit insulin-stimulated PI 3-kinase despite normal ty-
rosine phosphorylation of IRS-1 and binding of p85 regu-
latory subunit of PI 3-kinase (49). Another possibility is
that serine phosphorylation of the p85 subunit of PI
3-kinase is increased, which has been reported to decrease
the lipid kinase activity of the enzyme (50). In rats, injec-
tion of angiotensin II impaired insulin-mediated PI 3-ki-
nase activation via increased serine phosphorylation of
p85 without any change in the level of IR/IRS-1 tyrosine
phosphorylation (51,52). Furthermore, increased serine ki-
nase activity has been observed in insulin-resistant states
(53).
Intense interest has been focused on the identification
of those signaling steps downstream of PI 3-kinase that
may be implicated in glucose transport activation by
insulin. One of these is the serine-threonine kinase Akt,
which has been shown to be involved in the control of
glucose transport at a step downstream of PI 3-kinase (10).
However, it is still unclear whether insulin-stimulated Akt
activity is impaired in insulin-resistant skeletal muscle.
Insulin activation of Akt in the muscle of glucosamine-
infused rats (21), as well as in human diabetic subjects
(20), was found to be normal. However, insulin-stimulated
Akt in the muscle of type 2 diabetes subjects was reported
to be impaired at a maximal dose of insulin in a separate
study (23). In the present study, we found that insulin-
dependent Akt kinase activity is reduced in skeletal mus-
cle of high fat–fed rats, despite normal phosphorylation of
the enzyme on both Ser473 and Thr308. Whether this
alteration represents a primary defect leading to impaired
GLUT4 translocation in this animal model is still uncer-
tain. In L6 myoblasts, it has been shown that a Ͼ50%
decrease in Akt activity does not significantly affect
GLUT4 translocation (18). If such data could be extrapo-
lated to rat skeletal muscle, this would suggest that the
reduced Akt activity is unlikely to be responsible for the

lack of insulin action in muscle of high fat–fed animals.
Nevertheless, Akt kinase activity in insulin-stimulated
muscle of high fat–fed rats was reduced by as much as 40%
compared with insulin-stimulated chow-fed controls.
Thus, although Akt activation by insulin appears normal
based on Akt phosphorylation status, the actual ability of
the kinase to phosphorylate an exogenous substrate is
significantly altered. The mechanism behind this defect in
Akt kinase activity remains to be determined.
Another downstream target of PI 3-kinase that has been
suggested to be involved in the regulation of glucose trans-
port is the atypical member of the PKC family. It has been
shown to be activated by insulin in rat adipocytes (54),
3T3-L1 adipocytes, (19,55) and L6 myocytes (15). The use
of inhibitor (pseudosubstrate), transfection of kinase-inac-
tive or overexpression/constitutively active forms of the
kinase, as well as microinjection of antibodies against
aPKC, argued for its role in insulin-regulated glucose
transport (15,16,19,54–56). However, to the best of our
knowledge, there is as yet no experimental evidence for
insulin-dependent activation of aPKC in skeletal muscle,
the main target for insulin-stimulated glucose disposal. In
this study, we provide evidence that insulin stimulates
aPKC activity in skeletal muscle. This effect was demon-
strated by measurements of direct kinase activity as well
as by increased membrane association of aPKC. These
data are consistent with the proposition that aPKC is in-
volved in the stimulatory effect of insulin on glucose trans-
port in skeletal muscle. More importantly, we found that
aPKC was unresponsive to the action of insulin in skeletal
muscle of high fat–fed rats. The fact that insulin-induced
aPKC membrane association was also impaired in muscle
from insulin-infused (euglycemic-clamped) high fat–fed
animals further suggests that the lack of insulin action on
aPKC is sustained for at least 2 h of insulin exposure.
Impaired insulin-dependent activation of PI 3-kinase is
likely to explain part of the failure of insulin to activate
aPKC in rats fed the high-fat diet. A recent report by Kanoh
et al. (57) showed that activation of aPKC was resistant to
the action of insulin in adipocytes isolated from diabetic
animals, which was restored by thiazolidinedione treat-
ment with a concomitant increase in insulin-stimulated
glucose transport. The latter finding, together with our
results, supports a role for impaired aPKC signaling in
mediating insulin-resistant glucose transport in both adi-
pose tissue and skeletal muscle. Interestingly, we also
found that basal (non–insulin-stimulated) aPKC activity
and membrane localization are increased in high fat–fed
animals. It is not clear at the present time why aPKC
activity in the absence of insulin is abnormally elevated in
high fat–fed rats. This increase in aPKC activity was ob-
served even though basal glucose uptake was not affected
by high-fat feeding, although this may be explained by the
observation of decreased levels of both GLUT1 and GLUT4
in muscle from these animals. Whereas the higher insulin
levels in high fat–fed rats may partly explain the enhanced
basal aPKC activity, the fact that neither PI 3-kinase nor
Akt basal activities were increased in the same muscles
suggest that hyperinsulinemia is unlikely to be the main
factor involved. Another possibility is that other factors in
high fat–fed rats could have elevated muscle aPKC activ-
ity. Indeed, aPKCs are involved in many cellular processes,
and it is therefore possible that only a small fraction of the
total pool of aPKCs participates in the stimulation of
glucose transport. Among other roles of aPKC in cellular
signaling, it has been shown to participate in tumor necro-
sis factor-␣ (TNF-␣)-induced formation of ceramides by
sphingomyelinase (58,59). Although muscle TNF-␣ levels
were found to be similar between both dietary groups
(0.018 Ϯ 0.02 vs. 0.020 Ϯ 0.02 pg/␮g protein for chow- and
high fat–fed rats, respectively), we confirmed previous
reports of overexpression of the cytokine in white adipose
tissue of obese animals (0.91 Ϯ 0.35 vs. 1.76 Ϯ 0.36 pg/␮g
DNA for chow- and high fat–fed rats, respectively; P Ͻ
0.05) (60). It may therefore be speculated that local
production of TNF-␣ by surrounding adipose tissue in-
creases basal aPKC activity in muscle and subsequently
makes it unresponsive to the action of insulin in high
fat–fed rats.
In summary, the present study provides an extensive
characterization of the insulin signal transduction pathway
in skeletal muscle of the high fat–fed rat model of insulin
resistance. These animals showed a complete absence of
GLUT4 translocation in response to insulin not only to the
plasma membrane but also to the T-tubules, the major
component the muscle cell surface. We identified PI 3-
kinase as the first step of the insulin signaling pathway to
be altered by fat feeding. This was associated with re-
duced Akt kinase activity in insulin-stimulated muscle,
despite normal Akt phosphorylation. Moreover, we char-
acterized for the first time the insulin-dependent activation
and translocation of aPKC in normal and insulin-resistant
skeletal muscle. We found a complete failure of insulin to
activate aPKC in high fat–fed animals. Thus, alterations in
both Akt and aPKC signaling may be involved in the PI
3-kinase–dependent impairment in GLUT4 translocation
in the skeletal muscle of high fat–fed rats.
ACKNOWLEDGMENTS
This work was supported by grants from the Canadian
Diabetes Association (H.J. and A.M.). A.M. was supported
by scholarships from the Medical Research Council of
Canada and the Fonds de la Recherche en Sante´ du
Que´bec. F.T. was supported by a studentship from the
Que´bec Hypertension Society.
We thank Luce Dombrowski for expert technical assis-
tance. We are grateful to Romel Somwar, Dr. Philip Bilan,
and Dr. Amira Klip for their helpful advice on the PI
3-kinase assay and for critical reading of the manuscript.
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