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Two phases of palmitate-induced insulin resistance in skeletal muscle:

Impaired GLUT4 translocation is followed by a reduced GLUT4 intrinsic activity

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Two phases of palmitate-induced insulin resistance in skeletal muscle:
impaired GLUT4 translocation is followed by a reduced GLUT4
intrinsic activity
Hakam Alkhateeb,1 Adrian Chabowski,2 Jan F. C. Glatz,3 Joost F. P. Luiken,3 and Arend Bonen1
1
Department of Human Health and Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada; 2Department
of Physiology, Medical University of Bialystok, Bialystok, Poland; and 3Department of Molecular Genetics, Maastricht
University, Maastricht, The Netherlands
Submitted 15 December 2006; accepted in final form 4 June 2007
Alkhateeb H, Chabowski A, Glatz JF, Luiken JF, Bonen A. Two
phases of palmitate-induced insulin resistance in skeletal muscle: im-
paired GLUT4 translocation is followed by a reduced GLUT4 intrinsic
activity. Am J Physiol Endocrinol Metab 293: E783–E793, 2007. First
published June 5, 2007; doi:10.1152/ajpendo.00685.2006.—We exam-
ined, in soleus muscle, the effects of prolonged palmitate exposure (0,
6, 12, 18 h) on insulin-stimulated glucose transport, intramuscular
lipid accumulation and oxidation, activation of selected insulin-sig-
naling proteins, and the insulin-stimulated translocation of GLUT4.
Insulin-stimulated glucose transport was progressively reduced after
6 h (⫺33%), 12 h (⫺66%), and 18 h (⫺89%) of palmitate exposure.
These decrements were closely associated with concurrent reductions
in palmitate oxidation at 6 h (⫺40%), 12 h (⫺60%), and 18 h
(⫺67%). In contrast, intramuscular ceramide (⫹24%) and diacylglyc-
erol (⫹32%) concentrations, insulin-stimulated AS160 (⫺36%) and
PRAS40 (⫺33%) phosphorylations, and Akt (⫺40%), PKC␪
(⫺50%), and GLUT4 translocation (⫺40%) to the plasma membrane
were all maximally altered within the first 6 h of palmitate treatment.
No further changes were observed in any of these parameters after 12
and 18 h of palmitate exposure. Thus, the intrinsic activity of GLUT4
was markedly reduced after 12 and 18 h of palmitate treatment.
During this reduced GLUT4 intrinsic activity phase at 12 and 18 h, the
reduction in glucose transport was twofold greater compared with the
early phase (ⱕ6 h), when only GLUT4 translocation was impaired.
Our study indicates that palmitate-induced insulin resistance is pro-
voked by two distinct mechanisms: 1) an early phase (ⱕ6 h), during
which lipid-mediated impairments in insulin signaling and GLUT4
translocation reduce insulin-stimulated glucose transport, followed by
2) a later phase (12 and 18 h), during which the intrinsic activity of
GLUT4 is markedly reduced independently of any further alterations
in intramuscular lipid accumulation, insulin signaling and GLUT4
translocation.
glucose transport; glucose transporter 4; ceramide; diacylglycerol;
Akt; AS160; protein kinase C␨/␭; protein kinase C␪; palmitate oxi-
dation
IN INSULIN-SENSITIVE TISSUES, such as skeletal muscle, insulin
activates a signaling cascade that induces the translocation of
the glucose transporter GLUT4 from its intracellular depot(s)
to the cell surface, where these transport proteins, dock, fuse,
and become functionally active, thereby facilitating the trans-
port of glucose into the cell (for review see Ref. 61). In recent
years, dysregulated lipid metabolism has been associated with
inducing insulin resistance. Infusion with fatty acids induces
insulin resistance (5, 18, 42, 67), and there is a negative
Address for reprint requests and other correspondence: A. Bonen,Dept. of
Human Health and Nutritional Sciences, Univ. of Guelph, Guelph, ON N1G
2W1, Canada (e-mail: abonen@uoguelph.ca).
http://www.ajpendo.org 0193-1849/07 $8.00 Copyright © 2007 the American Physiological Society
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Am J Physiol Endocrinol Metab 293: E783–E793, 2007.
First published June 5, 2007; doi:10.1152/ajpendo.00685.2006.
relationship between insulin-stimulated glucose uptake and
intramuscular lipid accumulation, including tri- and diacylg-
lycerols, long-chain fatty acyl-CoAs, and ceramides (12, 17,
31, 49). It has also been speculated that a reduction in skeletal
muscle fatty acid oxidation contributes to the intracellular lipid
accumulation, thereby inducing insulin resistance (36).
Impairments in the postreceptor insulin-signaling pathway
are thought to be central in the development of fatty acid-
induced insulin resistance. Fatty acid infusion (4 –90 h) im-
paired the insulin-stimulated tyrosine phosphorylation of the
insulin receptor, insulin receptor substrate (IRS) ⫺1, and the
activities of PI 3-kinase, IRS-1-associated PI 3-kinase (5, 18,
35, 42, 67), and atypical PKC␨/␭ (38). Paradoxically, insulin
resistance in skeletal muscle provoked by lipid (38, 42) or
growth hormone infusion (32), as well as improved insulin
sensitivity provoked by thiazolidinedione treatment in type 2
diabetics (33, 39), failed to alter insulin-stimulated Akt phos-
phorylation (32, 34, 38, 39, 42) and AS160 phosphorylation
(34), downstream signals of PI 3-kinase. In high-fat-fed rats,
complete inhibition of insulin-stimulated GLUT4 translocation
in muscle only partially inhibited (⫺40%) insulin-stimulated
glucose transport (62). Collectively, these findings illustrate
that, although insulin sensitivity in muscle can be altered, the
mechanisms involved are not completely understood. Mecha-
nisms other than impaired insulin signaling may also contribute
to lipid-induced insulin resistance.
To stimulate glucose transport, insulin appears to activate
two mechanisms, 1) the well-known insulin-induced GLUT4
translocation from intracellular pools to the plasma membrane
(61) and 2) an increased intrinsic activity of cell surface
GLUT4 (3, 27, 30). In L6 muscle cells, insulin-stimulated
glucose transport can be reduced by altering the intrinsic
activity of cell surface GLUT4 in the absence of GLUT4
translocation and Akt phosphorylation (44, 59). Similarly, in
rat skeletal muscle, insulin-stimulated glucose transport rates
can be altered independently of the cell surface GLUT4 (11,
25, 62). Indeed, we have shown that epinephrine reduced
insulin-stimulated glucose transport in a dose-dependent man-
ner, whereas plasmalemmal GLUT4 remained unaltered (25).
In addition, high-fat feeding appears to lower the intrinsic
activity of GLUT4 (62). Thus, there is considerable evidence to
suggest that the activity of cell surface GLUT4 can be regu-
lated, a process that is not associated with the activation of p38
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in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
E783
E784 PALMITATE-INDUCED IMPAIRED GLUT4 TRANSLOCATION AND ACTIVITY
MAP kinase (3, 4, 63), as had been thought for some years. To
date, there appear to be no studies, either in muscle cells or in
mammalian tissues, that have examined whether fatty acids can
impair glucose transport by interfering with GLUT4 activity at
the cell surface.
Since the mechanisms contributing to fatty acid-induced
insulin resistance are not fully known, we have examined at
selected time intervals (0, 6, 12 and 18 h), in isolated mam-
malian muscle (soleus), the effects of palmitate on 1) insulin-
stimulated glucose transport, 2) intramuscular lipid accumula-
tion and oxidation, 3) activation of selected insulin signaling
proteins, and 4) the insulin-stimulated translocation of GLUT4.
Our studies have revealed that palmitate induces insulin resis-
tance in two ways: 1) during an early phase (ⱕ6 h), when
intracellular diacylglycerol (⫹32%) and ceramide (⫹24%)
accumulate and likely impair insulin-stimulated glucose trans-
port (⫺33%), in association with reductions in insulin-stimu-
lated Akt activation (⫺55%) and GLUT4 translocation
(⫺40%); 2) these events are followed by a subsequent later
phase at 12 and 18 h, during which, independently of any
further alterations in GLUT4 translocation, insulin signaling,
and intramuscular lipid accumulation, the intrinsic activity of
GLUT4 is markedly reduced.
METHODS
Materials
[1-14C]palmitate was purchased from Amersham Life Science
(Oakville, ON, Canada). Collagenase type II was purchased from
Worthington (Lakewood, NJ). Insulin (Humulin-R) was purchased
from Eli-Lilly (Toronto, ON, Canada). Penicillin and streptomycin
were purchased from Invitrogen (Grand island, NY). Silica plates (no.
60, 0.25 mm) were obtained from Merck KGaA (Darmstadt, Ger-
many). Total and phosphorylated proteins were determined with
commercially available antibodies from the following sources: anti-
Akt1/2, anti-Akt2, anti-phospho-Akt Ser473, and anti-phospho-Akt
Thr308 from Santa Cruz Biotechnology (Santa Cruz, CA); anti-
PKC␨/␭ and anti-PKC␪ from Santa Cruz Biotechnology (Santa Cruz,
CA); anti-IRS-1, anti-PI 3-kinase, anti-AS160, and anti-phospho-
AS160 (Thr642) from Upstate (Lake Placid, NY); anti-GLUT4 from
Chemicon International (Temecula, CA); goat-anti-rabbit secondary
antibodies from Chemicon International; and donkey-anti-rabbit
secondary antibody from Amersham Biosciences (Oakville, ON,
Canada). All other reagents were obtained from Sigma-Aldrich
(St. Louis, MO).
Animals
All experiments were approved by the Committee on Animal Care
at the University of Guelph. Male Sprague-Dawley rats (55–75 g),
bred on site, were used in these studies. The animals consumed
normal laboratory chow and water ad libitum. At the onset of each
experiment, rats were anesthetized with Somnotol (6 mg/100 g body
wt ip), and the soleus muscles were gently dissected free.
Muscle Incubation
We (2) have shown that soleus muscles (⬃20 mg) remain viable,
ex vivo, for up to 18 h. In the present study, intact soleus muscles
(⬃20 mg), after a 30 min preincubation, were incubated with (2 mM)
or without palmitate (control) for 0, 6, 12, or 18 h. At t ⫽ 0 min,
palmitate-treated muscles were exposed only briefly (5 min) to palmi-
tate, and control muscles were also briefly (5 min) incubated without
palmitate. All incubations (0 –18 h) were performed in 10 ml of
warmed (30°C), pregassed (95% O2-5% CO2) Medium 199 contain-
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ing 5 mM glucose supplemented with 4% bovine serum albumin V
(BSA), penicillin (100 IU/ml), and streptomycin (0.1 mg/ml). Low
concentrations of insulin (14.3 ␮U/ml) were also included, as failure
to maintain low concentrations of this hormone during a prolonged
incubation period reduces intramuscular phosphagens and glycogen
(65). The low concentrations of insulin did not stimulate glucose
transport (present study 14.3 ␮U/ml; data not shown). Incubation vials
were shaken at 110 cycles/min, and the gas phase and temperature
were maintained at 95% O2-5% CO2 and 30°C, respectively, through-
out. The incubation medium was replenished every 6 h.
ATP and Phosphocreatine
To ascertain the viability of incubated muscles, ATP and phospho-
creatine (PCr) concentrations were determined in muscles incubated
with and without palmitate for up to 18 h, as we (2) have described in
detail elsewhere.
Glucose Transport
At selected periods (0, 6, 12, and 18 h) insulin-stimulated glucose
transport was determined. Standard procedures were employed as we
have described previously (7, 9). Briefly, after the specified incubation
periods, soleus muscles were incubated (30°C, 30 min, 95% O2-5%
CO2) in 2 ml of palmitate-free Krebs-Henseleit buffer [8 mM glucose,
32 mM mannitol, and 0.1% BSA with or without insulin (20,000
␮U/ml)]. Subsequently, muscles were washed [2 ⫻ 10 min, 30°C,
glucose-free Krebs-Henseleit buffer, 40 mM mannitol, 0.1% BSA ⫾
insulin (20,000 ␮U/ml)]. Glucose transport was then determined in
palmitate-free Krebs-Henseleit buffer (2 ml) supplemented with 0.5
␮Ci 3-O-methyl-D-[3H]glucose (3-OMG), 1.0 ␮Ci [14C]mannitol, 32
mM 3-OMG, 4 mM mannitol, 4 mM pyruvate, and 0.1% BSA in the
presence (20,000 ␮U/ml) or absence of insulin for 20 min. Thereafter,
muscles were blotted, weighed, and solubilized followed by scintil-
lation counting of muscle extracts.
Palmitate Oxidation
To determine the rates of fatty acid oxidation, our previously
described method was used (19). Briefly, at the end of the incubation
periods (0, 6, 12 and 18 h), isolated soleus muscles that had been
incubated with and without palmitate for 0, 6, 12, and 18 h were
transferred to other glass vials containing 2 ml of pregassed (95%
O2-5% CO2) Medium 199 supplemented with 4% BSA and palmitate
(2 mM, 0.5 ␮Ci/ml [1-14C]palmitate; Amersham Life Science
Oakville, ON, Canada). Palmitate oxidation occurred at 30°C for 40
min, and the 14CO2 released was captured in a benzothium hydroxide
trap (400 ␮l, 1.0 M) In addition, at the end of the 40-min incubation
period, dissolved CO2 was released by adding sulfuric acid (1.0 ml,
1 M) to a 1.0-ml aliquot of the incubating medium and trapping the
14
CO2 in a benzothium hydroxide trap. Finally, water-soluble
14
C-labeled intermediates were extracted from muscles homogenized
after their incubation (19). After scintillation counting, the palmitate
oxidation rate was determined as we have done previously (19), by
summing the three sources of metabolized [14C]palmitate.
Intramuscular Triacylglycerol, Phospholipid, Diacylglycerol,
and Ceramide Concentrations
The intramuscular lipids were determined in muscles incubated in
Medium 199 with or without palmitate. At 0, 6, 12, and 18 h, muscles
were freeze-clamped and stored at ⫺80°C. To obtain sufficient mus-
cle, five soleii were pooled for each independent determination.
Intramuscular lipids [triacylglycerol (TAG), diacylglycerol (DAG),
phospholipids, ceramide] were extracted by the method of Folch et al.
(20), as modified by van der Vusse et al. (64). Lipids were extracted
from pulverized fat-free muscle samples in methanol (2 ml) and
chloroform (4 ml) containing the antioxidant butylated hydroxytolu-
ene (0.01%), and an internal standard (heptadecanoic acid) was added.
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 PALMITATE-INDUCED IMPAIRED GLUT4 TRANSLOCATION AND ACTIVITY
Water (1.5 ml) was added to the extracting mixture. One portion of the
chloroform layer was used to separate TAG, DAG, phospholipids, and
lipid fractions, and the second portion was used for ceramide deter-
minations. Thin-layer chromatography (silica plate no. 60, 0.25 mm;
Merck, Darmstadt, Germany) was used to separate lipids. For TAG,
DAG, and phospholipid separation a heptane-isopropyl ether-acetic
acid (60:40:3, vol/vol/vol) resolving solution (54) was used, and for
ceramide separation diethyl ether-hexane-acetic acid (90:10:1 vol/vol/
vol) was used as described by Yano et al. (66). After being resolved
and dried, lipid bands were visualized by spraying with a 0.2%
solution of 2⬘7⬘-dichlorofluorescein in methanol and identified under
UV light according to the standards on the plates. Each of the
separated lipids was methylated (45, 66) in 14% boron trifluoride-
methanol (14% BF3), and fatty acid methyl esters were extracted with
pentane (64). Finally, samples were dissolved in hexane and analyzed
by gas-liquid chromatography [Hewlett-Packard 5890 Series II gas
chromatograph with Varian CP-SIL capillary column (50 m ⫻ 0.25
mm internal diameter)] and flame-ionization detector (Agilent Tech-
nologies, Santa Clara, CA). The oven temperature was programmed
from 160°C to 225°C at 5°C/min and held at 225°C for 10 min.
According to the retention times of standards, the individual long-
chain fatty acids were quantified. The concentration of each lipid was
obtained by summing the fatty acids in their chromatograph profile.
E785
RESULTS
The selection of the palmitate concentration in the present
study was determined from dose response studies in which we
examined the effects of palmitate on the inhibition of insulin-
stimulated glucose transport after 12 h (Fig. 1A). Incubation
with 1.5–2 mM palmitate induced the maximal effects. At a
concentration of 2 mM, palmitate muscles remained metabol-
ically viable for up to 18 h, as shown by the constancy of the
ATP and PCr concentrations in all muscles (Fig. 1B).
Effect of Palmitate on Basal and Insulin-Stimulated Glucose
Transport in Soleus Muscles
In the absence of palmitate, basal 3-OMG transport rates
were not altered during the 18-h incubation period (P ⬎ 0.05;
Fig. 2). In the palmitate-treated muscles, the basal rates of

Plasma Membrane Preparation

The soleus muscle plasmalemmal content of selected proteins was
determined in muscles incubated in Medium 199 for 0, 6, 12, and 18 h
with or without palmitate. At these selected time points, the muscles
were treated with (20,000 ␮U/ml) or without insulin for 70 min to
mimic the time course in the foregoing glucose transport experiments.
To obtain sufficient plasma membrane, 10 incubated soleii were
pooled for each independent experiment. Giant vesicle plasma mem-
branes were obtained as we have previously reported in detail (8, 10).
In separate experiments, we established that insulin stimulated the
appearance of GLUT4, Akt, and PKC␪ and PKC␨/␭ at the plasma
membrane.

Protein Analysis
The soleus muscle total protein of selected proteins was determined
in muscles incubated in Medium 199 for 0, 6, 12, and 18 h with or
without palmitate. Thereafter, muscles were rapidly blotted, frozen in
liquid nitrogen, and stored at ⫺80°C until analyzed for selected
proteins. To measure the phosphorylation status of Akt, PRAS40 (a
proline-rich Akt substrate), and AS160, muscles were incubated for 0,
6, 12, and 18 h with and without palmitate followed by incubation in
palmitate-free Krebs-Henseleit buffer in the presence of insulin
(20,000 ␮U/ml) for 10 min, the time in which maximal phosphory-
lation was observed (H. Alkhateeb and A. Bonen, unpublished data,
and Ref. 57). Thereafter, the muscles were rapidly blotted, frozen, and
stored at ⫺80°C for later analysis.

Muscle Protein Extraction and Western Blotting

For whole muscle protein determination, two frozen soleii were
homogenized in 2 ml of buffer (8, 10). Muscle homogenate and
plasma membrane protein concentrations were determined using the
bicinchoninic acid assay. Proteins were separated using SDS-poly-
acrylamide gel electrophoresis and were detected using Western
blotting. We (2, 8, 10) have reported these procedures previously.
Statistics
Data were analyzed using two-way analyses of variance, and when
appropriate, a Fisher’s least significant difference post hoc analysis
was used. For some small experiments, the data were analyzed with a
one-way analysis of variance when this was warranted by the exper-
imental design. All data are reported as means ⫾ SE.
Fig. 1. Dose-response relationship of palmitate on insulin-stimulated 3-O-
methylglucose (3-OMG) transport in soleus muscle (A) and ATP and phos-
phocreatine (PCr) concentrations in isolated soleus muscle incubated for 0, 6,
12, and 18 h in the absence and presence of 2 mM palmitate (B). Data are total
3-OMG transport (basal ⫹ insulin-stimulated) and are presented as means ⫾
SE; n ⫽ 4 –5 muscles per data point. In some cases error bars are less than the
plot symbol. For both A and B, at t ⫽ 0, control muscles were briefly incubated,
while palmitate-treated muscles were briefly exposed to palmitate (see METH-
ODS). A: incubations without (0 mM) or with palmitate (0.5–2.0 mM) occurred
for 12 h, after which muscles were exposed to insulin. *P ⬍ 0.05, 0.5 vs. 0
mM; **P ⬍ 0.05, 1.0 vs. 0.5 mM; ***P ⬍ 0.05, 1.5 vs. 1.0 mM, and 2.0 vs
1.0 mM (asterisks denote comparisons based on post hoc tests). B: n ⫽ 8 –10
muscles per data point; there were no significant differences at any point.

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E786 PALMITATE-INDUCED IMPAIRED GLUT4 TRANSLOCATION AND ACTIVITY

treatment did not further reduce the insulin-stimulated GLUT4

content at the plasma membrane at 12 and 18 h (P ⬎ 0.05; Fig.
4B), despite the continued reduction in insulin-stimulated glu-
cose transport (Fig. 2).
At 6 h, the concurrent decrease in 3-OMG transport and
plasma membrane GLUT4 in the palmitate-treated muscles
yielded a ratio (Fig. 4C) that did not differ from that at t ⫽ 0,
before the onset of insulin resistance. However, after 6 h, there

Fig. 2. Effect of prolonged palmitate exposure (0 –18 h) on basal and insulin-

stimulated 3-OMG transport in soleus muscle. At t ⫽ 0, control muscles were
briefly incubated, while palmitate-treated muscles were briefly exposed to
palmitate (see methods). Data are presented as means ⫾ SE; n ⫽ 8 muscles per
data point. In some cases error bars are less than the plot symbol. Note that
insulin was present for only 70 min (see METHODS) after 0, 6, 12, or 18 h of
muscle incubation. Control: no differences among time points (P ⬎ 0.05).
Palmitate treatment: *P ⬍ 0.05, 6 vs. 0 h; **P ⬍ 0.05, 12 vs. 6 h; ***P ⬍ 0.05,
18 vs. 12 h (asterisks denote comparisons based on post hoc tests).

3-OMG transport were reduced only in the first 6 h (P ⬍ 0.05).

Thereafter (6 –18 h), basal 3-OMG transport remained unal-
tered in palmitate-treated muscles (P ⬎ 0.05; Fig. 2).
Insulin-stimulated glucose transport was not altered in the
control muscles (P ⬎ 0.05; Fig. 2). In contrast, palmitate
induced a very marked, progressive reduction in insulin-stim-
ulated 3-OMG transport over 18 h. After 6, 12, and 18 h,
glucose transport was reduced by 33% (P ⬍ 0.05), 66% (P ⬍
0.05), and 89% (P ⬍ 0.05), respectively, relative to t ⫽ 0 h.
Notably, after 18 h of palmitate exposure, insulin-stimulated
glucose transport did not differ from basal glucose transport at
t ⫽ 0 h (P ⬎ 0.05; Fig. 2).
Effects of Insulin on Plasmalemmal GLUT4, Akt, and PKC␪
and PKC␨/␭
Although it had been reported some years ago that insulin-
stimulated GLUT4 appearance at the plasma membrane of
giant vesicles could not be detected (50), we found that insulin
increased plasmalemmal GLUT4 (2.3-fold, P ⬍ 0.05; Fig. 3). In
addition, insulin also increased plasmalemmal Akt (1.5-fold),
PKC␪ (3.3-fold), and PKC␨/␭ (4.8-fold, P ⬍ 0.05; Fig. 3).
Effect of Palmitate on Protein Expression of GLUT4
and Insulin-Stimulated Plasmalemmal GLUT4
The expression of GLUT4 protein was not altered during the
18-h incubation period in the control and palmitate-treated
muscles, (P ⬎ 0.05; Fig. 4A). Basal levels of plasma membrane
GLUT4 were also not altered during the 18-h incubation period
in either the control or palmitate-treated muscles, (P ⬎ 0.05;
Fig. 4B).
In control muscles, insulin induced a 2.5-fold increase in Fig. 3. Effects of insulin on plasma membrane GLUT4, Akt, PKC␪, and
plasmalemmal GLUT4 at the end of each of the incubation PKC␨/␭. Data are presented as means ⫾ SE; GLUT4, n ⫽ 9 independent
periods (0, 6, 12, and 18 h, P ⬍ 0.05; Fig. 4B). The insulin- determinations; Akt, n ⫽ 3 independent determinations; PKC␪, n ⫽ 4 inde-
stimulated plasmalemmal GLUT4 content was not altered pendent determinations; PKC␨/␭, n ⫽ 4 independent determinations. To obtain
throughout the 18-h period in the control muscles (P ⬎ 0.05; sufficient plasma membrane for each independent determination, 10 solei (⬃20
mg each) were incubated with or without insulin for 30 min. These 10 muscles
Fig. 4B). In contrast, in palmitate-treated muscles, the insulin- were then pooled for each treatment (basal and insulin stimulation) to obtain
stimulated plasmalemmal GLUT4 was decreased by 40% sufficient giant vesicles plasma membrane for Western blotting. *P ⬍ 0.05,
within the first 6 h (P ⬍ 0.05; Fig. 4B). Thereafter, palmitate insulin vs. basal.

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 PALMITATE-INDUCED IMPAIRED GLUT4 TRANSLOCATION AND ACTIVITY
was a sharp progressive decrease in the ratio of 3-OMG
transport to plasma membrane GLUT4 in the palmitate treated
muscles (Fig. 4C).
Effect of Palmitate on Expression and Insulin-Stimulated
Phosphorylation of Signaling Proteins
The protein expressions of IRS-1, PI 3-kinase, Akt1/2, Akt2,
AS160, PRAS40, PKC␨/␭, and PKC␪ remained unchanged
during the 18-h incubation period in either presence or absence
of palmitate (n ⫽ 5–7 muscles at all time points in control and
palmitate-treated muscles, P ⬎ 0.05; data not shown). Under
basal conditions, the phosphorylation states of Akt (Thr308 and
Ser473) were not altered (P ⬎ 0.05; Fig. 5) in either control or
palmitate-treated muscles. Relative to t ⫽ 0, the basal phos-
phorylation states of AS160 and PRAS40 were reduced some-
what, and to a similar extent, in both control and palmitate-
treated muscles (Fig. 5, C and D).
In the control muscles, the insulin-induced phosphorylation
states of Akt Thr308 (Fig. 5A) and Ser473 (Fig. 5B) and AS160
(Fig. 5C) were not changed (P ⬎ 0.05) during the 18-h
incubation period. In contrast, PRAS40 phosphorylation, rela-
tive to t ⫽ 0 h, was increased after 12 h (⫹14%) and 18 h
(⫹27%) (P ⬍ 0.05; Fig. 5D). Palmitate did not alter the
insulin-induced phosphorylation of Akt Thr308 (Fig. 5A) and
Ser473 (Fig. 5B) at any time point (P ⬎ 0.05). In contrast,
palmitate treatment reduced the insulin-stimulated phosphory-
lation of AS160 in the first 6 h (⫺36%, P ⬍ 0.05; Fig. 5C), but
it was not changed further thereafter, as AS160 phosphoryla-
tion remained stably depressed at 12 h (⫺32%) and 18 h
(⫺40%) (Fig. 5C). A similar reduction pattern was observed
for insulin-stimulated phosphorylation of the proline-rich Akt
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E787
Fig. 4. Effect of prolonged palmitate exposure
(0 –18 h) on soleus muscle protein expressions
of GLUT4 (A), on basal plasma membrane
GLUT4 and insulin-stimulated plasma mem-
brane GLUT4 (B), and on the ratio of 3-OMG
transport to plasma membrane GLUT4 (C). At
t ⫽ 0, control muscles were briefly incubated,
while palmitate-treated muscles were briefly
exposed to palmitate (see METHODS). Data are
presented as means ⫾ SE. In some cases error
bars are less than the plot symbol. Note that
insulin was present for only 70 min (see METH-
ODS) after 0, 6, 12, or 18 h of muscle incuba-
tion. A: n ⫽ 5– 6 muscles per data point. B: n ⫽
7 independent experiments for each data point.
To obtain sufficient plasma membrane, 10 solei
were pooled for each independent experiment
at each time point. C: ratio was calculated from
means in insulin-stimulated muscles in Fig. 2
and here in B. Basal plasma membrane GLUT4
levels in control (P ⬎ 0.05) and palmitate
treated muscle (P ⬎ 0.05) were unaltered.
Insulin stimulation in control muscle resulted
in a 2.5-fold increase in plasma membrane
GLUT4 at 0 to 18 h, P ⬍ 0.05. Palmitate
treatment ⫹ insulin-stimulation: *P ⬍ 0.05 at
6, 12, and 18 h vs. control muscles at corre-
sponding time points (asterisks denote compar-
isons between control and palmitate treated
conditions based on post hoc tests). In palmi-
tate-treated muscle at 12 and 18 h, insulin-
stimulated plasma membrane GLUT4 does not
differ from basal plasma membrane GLUT4.
substrate PRAS40 (40), in palmitate-treated muscles (reduction
relative to t ⫽ 0 h: ⫺23% at 6 h, ⫺18% at 12 h, ⫺22% at 18 h;
Fig. 5D).
Effect of Palmitate on Insulin-Stimulated Plasmalemmal Akt,
PKC␨/␭, and PKC␪
Akt. In control muscles, during the 18-h incubation period
the insulin-stimulated plasmalemmal Akt was not altered in the
first 6 h (P ⬎ 0.05; Fig. 6A) but was increased thereafter at 12 h
(⫹55%, P ⬍ 0.05; Fig. 5A) and 18 h (⫹77%, P ⬍ 0.05; Fig.
6A). These increments in plasmalemmal Akt at 12 and 18 h did
not differ (P ⬍ 0.05; Fig. 6A). In contrast, palmitate treatment
was associated with an impaired insulin-stimulated Akt trans-
location to the plasma membrane within the first 6 h (⫺55%,
P ⬍ 0.05; Fig. 6A). Relative to the 6-h time point, this
insulin-stimulated translocation of Akt to the plasma mem-
brane had recovered slightly at 12 and 18 h (P ⬍ 0.05; Fig.
6A). However, relative to t ⫽ 0, insulin-stimulated transloca-
tion of Akt to the plasma membrane was still inhibited in
palmitate-treated muscles at 12 h (⫺32%, P ⬍ 0.05; Fig. 6A)
and at 18 h (⫺36%, P ⬍ 0.05; Fig. 6A).
PKC␨/␭. In the control muscles, the insulin-stimulated trans-
location of PKC␨/␭ to the plasma membrane was not altered
(P ⬎ 0.05; Fig. 6B). In the palmitate-treated muscles, insulin-
stimulated translocation of PKC␨/␭ to the plasma membrane
was also not altered, except for an increase at 12 h (⫹23%, P ⬍
0.05; Fig. 6B).
PKC␪. In the control muscles, there was a reduction in the
insulin-stimulated PKC␪ translocation to the plasma mem-
brane within the first 6 h (⫺20%, P ⬍ 0.05; Fig. 6C). This was
renormalized by 12 h, whereas after 18 h there was an increase
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E788 PALMITATE-INDUCED IMPAIRED GLUT4 TRANSLOCATION AND ACTIVITY

Fig. 5. Phosphorylation of Akt Thr308 (A), Akt

Ser473 (B), AS160 (C), and PRAS40 (a proline-rich
Akt substrate; D) in control and palmitate-treated
muscles under basal and insulin stimulated (10 min)
conditions. At t ⫽ 0, control muscles were briefly
incubated, while palmitate-treated muscles were
briefly exposed to palmitate (see METHODS). Data
are presented as means ⫾ SE; n ⫽ 3– 4 muscles for
basal conditions and n ⫽ 5–7 muscles for insulin
treatment for each data point. In some cases error
bars are less than the plot symbol. As figure is
already crowded, blots for basal phosphorylation
states are not shown. Note that insulin was present
for only 10 min (see METHODS) after 0, 6, 12, or 18 h
of muscle incubation. Note in D, the reversal of
⫹palmitate and ⫺palmitate loading compared with
A–C. In all instances, basal phosphorylations are
less than in muscles treated with insulin. In addi-
tion, basal control and basal palmitate-treated mus-
cles did not differ at any point except at 6 h in D.
Insulin treatment: *P ⬍ 0.05, 6, 12, or 18 h vs. 0 h;
**P ⬍ 0.05, 12 vs. 0 h; ***P ⬍ 0.05 18 vs. 12 h.
Basal: †P ⬍ 0.05, 12 vs. 0 h for control and
palmitate-treated; ††P ⬍ 0.05, 18 vs. 12 h, for
control and palmitate-treated; ⫹P ⬍ 0.05, 6 vs. 0 h
for control. Symbols denote comparisons based on
post hoc tests.

in the insulin-stimulated plasmalemmal PKC␪ (⫹50%, P ⬍
0.05; Fig. 6C). In contrast, in muscles that were treated with
palmitate, insulin-stimulated plasmalemmal PKC␪ was re-
duced at 6 h (⫺50%, P ⬍ 0.05; Fig. 6C), at 12 h (⫺45%, P ⬍
0.05; Fig. 6C) and at 18 h (⫺35%, P ⬍ 0.05;, Fig. 6C)
compared with insulin-stimulated PKC␪ translocation at t ⫽ 0.
Intramuscular Lipid Accumulation and Palmitate Oxidation
In control muscles, the concentrations of the intramuscular
lipids were relatively stable during the 18-h incubation. How-
ever, in control muscles, TAG concentrations were increased
somewhat at 18 h (P ⬍ 0.05; Fig. 7A). Phospholipid concen-
trations decreased somewhat (⫺22%) in both control and
palmitate-treated muscles over the 18-h period, with most of
the reduction (⫺15%) occurring during the last 6 h (P ⬍ 0.05,
data not shown). DAG (Fig. 7B) and ceramide (Fig. 7C)
concentrations were not altered in control muscles, except at
12 h when a reduction was observed in DAG (⫺19%, P ⬍
0.05; Fig. 7B).
Incubation with palmitate increased the intramuscular TAG
concentration, but only when incubated for more than 6 h with
palmitate (Fig. 7A). By 12 h, the intramuscular TAG concen-
tration had increased 53% (P ⬍ 0.05) and remained at that
level until 18 h (Fig. 7A). Palmitate incubation increased DAG
concentrations by 31% after 6 h (P ⬍ 0.05; Fig. 7B). After 12
and 18 h of incubation with palmitate, DAG concentrations
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were 11% (P ⫽ 0.075) and 19% (P ⬍ 0.05) greater, respec-
tively, than at t ⫽ 0 (Fig. 7B). Palmitate treatment also
increased the ceramide concentrations after 6 h (⫹24%, P ⬍
0.05; Fig. 7C) and relative to t ⫽ 0 these concentrations
remained elevated at that level at 12 h (⫹17%, P ⬍ 0.05; Fig.
7C) and at 18 h (⫹33%, P ⬍ 0.05; Fig. 7C).
The rates of palmitate (2 mM) oxidation were not altered in
control muscles (P ⬎ 0.05; Fig. 7D). In contrast, in the
palmitate-treated muscles, palmitate oxidation decreased pro-
gressively (P ⬍ 0.05; Fig. 7D), by 40% and 60% at 6 h and
12 h, respectively (P ⬍ 0.05; Fig. 6D). Relative to the 12-h
reduction, palmitate oxidation was not reduced any further at
18 h in the palmitate-treated muscles (P ⬎ 0.05; Fig. 7D).
Interrelationships Among 3-OMG Transport, Plasma
Membrane GLUT4, and Selected Variables
Many factors have been implicated in fatty acid-induced
impairments in insulin-stimulated glucose transport. Palmitate
treatment altered a number of parameters only within the first
6 h, with no further changes thereafter (i.e., DAG and ceramide
concentrations, insulin-stimulated phosphorylation of AS160
and PRAS40, and insulin-stimulated plasmalemmal Akt,
PKC␪, and GLUT4; Fig. 8A). However, these changes were
not closely associated with the progressively greater reduction
in insulin-stimulated glucose transport, particularly during the
6- to 18-h period of palmitate exposure. Among all parameters
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 PALMITATE-INDUCED IMPAIRED GLUT4 TRANSLOCATION AND ACTIVITY
examined over the 18-h period, there was a strong relationship
between the reductions in the rate of palmitate oxidation and
the rates of insulin-stimulated glucose transport in palmitate-
treated muscles (Fig. 8, B and C).
DISCUSSION
We have examined the mechanisms involved in palmi-
tate-induced insulin resistance in skeletal muscle over a
prolonged period of time (0, 6, 12, and 18 h). The key novel
finding is that palmitate provokes insulin resistance in
skeletal muscle by two separate mechanisms: one linked to
an intracellular, lipid-associated impairment in insulin sig-
naling and GLUT4 translocation and another that is associ-
ated with a reduced intrinsic activity of plasmalemmal
GLUT4, independent of further inhibition of insulin signal-
ing. Moreover, the disorders in insulin signaling and GLUT4
translocation precede the reduction in GLUT4 intrinsic activ-
ity. Compared with the impaired GLUT4 translocation, the
reduced intrinsic activity of GLUT4 accounted for a far greater
(2-fold) impairment in insulin-stimulated glucose transport.
The muscles in the present study, as in other long-term
incubation studies (2, 65), were metabolically viable and re-
mained insulin responsive. The use of a high palmitate con-
centration to induce insulin resistance is similar to previous
approaches [2 mM (60); 1.6 mM (48)].
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E789
Fig. 6. Soleus muscle plasma membrane Akt
(A), PKC␨/␭ (B), and PKC␪ (C) in control and
palmitate-treated muscles exposed to insulin. At
t ⫽ 0, control muscles were briefly incubated,
while palmitate-treated muscles were briefly ex-
posed to palmitate (see METHODS). Data are pre-
sented as means ⫾ SE; n ⫽ 5–7 independent
experiments for each data point. In some cases
error bars are less than the plot symbol. Note
that insulin was present for only 70 min (see
METHODS) after 0, 6, 12, or 18 h of muscle
incubation. To obtain sufficient plasma mem-
brane 10 solei were pooled for each independent
experiment. *P ⬍ 0.05 vs. 0 h; **P ⬍ 0.05,
control vs. palmitate-treated muscles; ***P ⬍
0.05 12 or 18 h vs. 6 h (asterisks denote com-
parisons based on post hoc tests).
Palmitate Impairs Insulin-Stimulated Glucose Transport
and GLUT4 Translocation and Activity
The palmitate-induced impairment in insulin-stimulated glu-
cose transport consisted of an early and a late phase. Each of
these appeared to be attributable to different mechanisms.
Early-phase (⬍6 h) impaired glucose transport and
GLUT4 translocation. The palmitate (2 mM)-induced reduc-
tion (⫺33%) in insulin-stimulated glucose transport after 6 h
parallels the observations in two other, similar studies, in
which palmitate also reduced insulin-stimulated glucose trans-
port in isolated soleus [⫺25% after 6 h with 2 mM palmitate
(60)] and extensor digitorum longus muscles [⫺22%, after 5 h
with 1.6 mM palmitate (48)]. These changes were not attrib-
utable to the minimal, palmitate-induced reduction in basal
glucose transport (present study and Ref. 48) or to an altered
GLUT4 protein expression (present study). Instead, the im-
paired insulin-stimulated GLUT4 translocation (⫺44%) ap-
peared to account for the similar reductions in glucose trans-
port (⫺33%) within the first 6 h. This is also suggested by the
similar ratio of insulin-stimulated glucose transport to plasma
membrane GLUT4 in the 6-h palmitate-treated muscles com-
pared with the control muscle ratios at every time point (see
Fig. 4C).
Late-phase impaired insulin-stimulated glucose transport
and GLUT4 activity. With prolonged palmitate incubation
(ⱕ18 h), the insulin-stimulated glucose transport was reduced
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E790 PALMITATE-INDUCED IMPAIRED GLUT4 TRANSLOCATION AND ACTIVITY

Fig. 7. Effects of prolonged palmitate exposure

(0 –18 h) on intramuscular concentrations of
triacylglycerol (A), diacylglycerol (B), and cer-
amide (C), and palmitate oxidation (D) in soleus
muscle. Data are presented as means ⫾ SE; for
A–C, n ⫽ 5 independent experiments, each
independent observation consisting of 5 pooled
solei; for D, n ⫽ 8 muscles per data point. In
some cases error bars are less than the plot
symbol. At t ⫽ 0, control muscles were briefly
incubated, while palmitate-treated muscles were
briefly exposed to palmitate (see METHODS).
*P ⬍ 0.05, 6 h vs. 0 h; **P ⬍ 0.05, 12 or 18 h
vs. 6 h; ***P ⬍ 0.05, control vs. palmitate-
treated (asterisks denote comparisons based on
post hoc tests).

progressively further at 12 h (⫺66%) and 18 h (⫺89%). These
reductions were not attributable either 1) to changes in GLUT4
protein expression or 2) to further reductions in the insulin-
stimulated GLUT4 translocation to the plasma membrane.
Thus, the late-phase, twofold greater insulin resistance was
associated with a reduced GLUT4 intrinsic activity. We rec-
ognize that preparative procedures designed to isolate plasma
membrane fractions may not be fully representative of the
plasma membrane in intact muscle in which the glucose trans-
port rates were determined. Nevertheless, our study strongly

Fig. 8. Relationship between the relative (%)

changes in intramuscular lipids, insulin-stimulated
AS160 phosphorylation (A), and insulin-stimulated
plasma membrane PKC␪, Akt, and GLUT4 in 0- to
18-h palmitate-treated soleus muscles (B) between
rates of insulin-stimulated glucose transport and
rates of palmitate oxidation, and between relative
(%) changes in insulin-stimulated glucose trans-
port and rates of palmitate oxidation (C). In B, a
nonlinear regression line was obtained by fitting
mean values of palmitate acid oxidation vs. insu-
lin-stimulated glucose transport, as these data were
obtained from different muscles. Means ⫾ SE
were from data obtained in palmitate-treated mus-
cles at 0, 6, 12, and 18 h and are shown in Figs. 2
and 7D.

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 PALMITATE-INDUCED IMPAIRED GLUT4 TRANSLOCATION AND ACTIVITY
suggests that a second, somewhat delayed mechanism is in-
volved in palmitate-induced insulin resistance, namely a re-
duced intrinsic activity of plasmalemmal GLUT4 (see Fig. 4C).
Changes In Intramuscular Lipids During Long-Term
Palmitate Incubation (0 –18 h)
Fatty acids have been widely implicated in the induction of
insulin resistance (present study and refs. 6, 31, 49). Our study
supports the idea that the intramuscular accumulation of tri-
acylglycerols per se does not induce insulin resistance, as their
accumulation occurred well after insulin resistance had been
observed. Ceramides and DAG are more likely to inhibit
insulin signaling (1, 16, 28, 43). In palmitate-treated muscles,
intramuscular ceramide and DAG accumulations coincided
with the onset of insulin resistance within the first 6 h. How-
ever, it appears that these metabolites are not directly involved
with the late-phase (12 and 18 h), more severe insulin resis-
tance that developed, since the intramuscular ceramide and
DAG accumulations were not increased further at 12 and 18 h.
The reduced rate of palmitate oxidation in the palmitate-
treated muscles corresponded most closely with the reduced
rate of insulin-stimulated glucose transport during both the
early (ⱕ6 h) and late phases (12 and 18 h). The reason for this
is unclear; however, others (36) have argued that the impaired
fatty acid oxidation is closely associated with skeletal muscle
insulin resistance.
Palmitate-Mediated Inhibition Of Insulin Signaling
Impairments in the post-receptor insulin-signaling pathway
are thought to be central in the development of fatty acid-
induced insulin resistance. (5, 18, 26, 35, 38, 41, 42, 47, 62,
67). In the present study, palmitate treatment impaired the
insulin-induced activation of Akt, PRAS40, AS160, and PKC␪
t PKC␨/␭. However, these impairments were all observed
within the first 6 h, with no further impairment thereafter at 12
and18 h. Therefore, we focus our discussion on the changes in
the early-phase (ⱕ6 h) impairments in insulin signaling.
Early-Phase (ⱕ6 H) Lipid-Mediated Impairment in Akt
There is considerable disagreement as to whether fatty acids
impair insulin-stimulated glucose transport via a reduction in
Akt phosphorylation (13–15, 22, 38, 42, 52, 56, 58, 60).
Although insulin effects on Akt Ser473 and Thr308 phosphory-
lations were not altered by palmitate treatment, it did appear
that palmitate contributed to decreasing Akt activity, since
1) reductions occurred in the insulin-induced phosphoryla-
tions of AS160 and PRAS40 (downstream Akt targets), and
2) reductions occurred in the insulin-stimulated appearance of
Akt at the plasma membrane, as has been observed in palmi-
tate-treated L6 muscle cells (52). The different effects of
palmitate on insulin-stimulated Akt phosphorylation and Akt
translocation may be attributable to the discrepancies that can
arise between Akt Ser473 and Thr308 phosphorylation and Akt
activity (41, 62).
It is thought that ceramides, derived from saturated fatty
acids, interfere with insulin action by reducing Akt phosphor-
ylation/activation and translocation (14, 15, 24, 28, 51, 52, 56).
Therefore, in the palmitate-treated muscles, the ceramide ac-
cumulation (⫹24%) within the first 6 h likely accounted for the
impaired Akt activation. Presumably, this resulted in the im-
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E791
paired AS160 phosphorylation, which then inhibited GLUT4
translocation. However, this sequence of actions cannot ac-
count for the further reductions in insulin-stimulated glucose
transport observed at 12 and 18 h, as neither Akt activation nor
GLUT4 translocation were changed beyond that observed
within the first 6 h.
Increased ceramide concentrations that occur with lipid
infusion (38) or high-fat feeding (26, 41) can also contribute to
the inhibition of insulin-stimulated Akt activity via the activa-
tion of PKC␨/␭ (26, 38, 41), although this is not always
observed (62). In the present study, it appears that PKC␨/␭ was
not activated by ceramide accumulation, but the absence of a
direct measurement of PKC enzyme activity precludes a de-
finitive conclusion on this matter. Nevertheless, it is known
that ceramide-induced PKC␨/␭ activation is not the only means
for inhibiting Akt activity, as this can be inhibited by an
alternative, ceramide-mediated mechanism, namely the activa-
tion of a type 2A-like phosphatase, which plays a role in the
dephosphorylation of Akt (13).
It has been proposed that DAG accumulation induces insulin
resistance via the activation of PKC␪ (23, 31, 67), which
antagonizes insulin signaling by increasing serine phosphory-
lation of IRS-1 (23, 67). Moreover, PKC␪-null mice do not
develop insulin resistance when placed on a high-fat diet (37).
Although an increase in plasmalemmal PKC␪ is indicative of
its activation (55), in the present study the insulin-stimulated
appearance of PKC␪ was not altered. This suggests that insulin
resistance in the palmitate-treated muscles was not associated
with DAG-mediated activation of PKC␪.
Late-Phase (12 And 18 h) Impaired Plasmalemmal
GLUT4 Activity
The data in the present study suggest strongly that pro-
longed palmitate treatment (⬎6 h) interfered with insulin
action by mechanisms that are independent of 1) altered
insulin signaling and 2) impaired GLUT4 translocation to
the plasma membrane. One of these mechanisms may be the
reduction of the intrinsic activity of the plasmalemmal
GLUT4.
In early studies in skeletal muscle (11, 21), a discrepancy
was found between the changes in cell surface GLUT4 and
rates of glucose transport. Therefore, it was proposed that
glucose transport was regulated via both GLUT4 translocation
and intrinsic activity. However, this concept fell into disfavor
for a number of years, only to be revived in recent years (3, 25,
27, 29, 30, 46). We (25) have shown that epinephrine induces
insulin resistance in skeletal muscle in a dose-dependent man-
ner by inhibiting GLUT4 activity. The present study also
supports the idea that GLUT4 intrinsic activity can be altered.
The mechanism by which the intrinsic activity of cell surface
GLUT4 is altered remains obscure. Contrary to some earlier
studies, recent work has shown that p38 MAPK activation is
not involved in altering the intrinsic activity of GLUT4 (3, 4,
53, 63). Nevertheless, there is substantial evidence to indicate
that cell surface GLUT4 activity can be altered to regulate
glucose transport (3, 11, 21, 25, 27, 30, 53, 63). The present
study indicates that the palmitate-induced reduction in the
intrinsic activity of GLUT4 occurs after insulin-stimulated
GLUT4 translocation has been impaired.
293 • SEPTEMBER 2007 • www.ajpendo.org
E792 PALMITATE-INDUCED IMPAIRED GLUT4 TRANSLOCATION AND ACTIVITY
Summary
Our study has shown that palmitate-induced insulin resis-
tance is provoked by two distinct mechanisms. First, in the
early phase (⬍6 h), GLUT4 translocation is impaired, resulting
from the intramuscular accumulation of diacylglycerol and
ceramide and the concurrent impairments in insulin-stimulated
Akt, PRAS40, and AS160 activation. Second, another phase is
evident after 12 and 18 h of palmitate exposure, during which
the intrinsic activity of GLUT4 is markedly reduced. This later
phase is independent of any further alterations in GLUT4
translocation, insulin signaling, and intramuscular lipid accu-
mulation. Compared with the impaired GLUT4 translocation,
the reduced intrinsic activity of GLUT4 accounted for a far
greater (2-fold) impairment in insulin-stimulated glucose trans-
port.
GRANTS
Studies in our laboratories are supported by grants from the Canadian
Institutes of Health Research, the Natural Sciences and Engineering Research
Council of Canada, The Heart and Stroke Foundation of Ontario, the Nether-
lands Heart Foundation (2002T049), the European Community (Integrated
Project LSHM-CT-2004-005272, Exgenesis), KBN 3P05B 19022 (Poland),
and the Canada Research Chair program. H. Alkhateeb was supported by a
scholarship from The Hashemite University, Zarqa, Jordan. J. J. F. P. Luiken
is a recipient of a VIDI-Innovation Research Grant from the Netherlands
Organization for Scientific Research (NWO-ZonMw Grant 016.036.305). A.
Bonen is the Canada Research Chair in Metabolism and Health.
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