J Appl Physiol 100: 1467–1474, 2006.
First published December 15, 2005; doi:10.1152/japplphysiol.01438.2005.
Saturated, but not n-6 polyunsaturated, fatty acids induce insulin resistance:
role of intramuscular accumulation of lipid metabolites
Jong Sam Lee,1,* Srijan K. Pinnamaneni,3,* Su Ju Eo,2 In Ho Cho,2 Jae Hwan Pyo,2
Chang Keun Kim,2 Andrew J. Sinclair,4 Mark A. Febbraio,3 and Matthew J. Watt3
1
Department of Physical Therapy, College of Medicine, Eulji University, Young-Du Dong, Jung-Gu, Daejeon,
Human Physiology Laboratory, Korean National Sports University, Ohryun-Dong, Song-Pa Gu,
Seoul, South Korea; 3Cellular and Molecular Metabolism Laboratory, School of Medical Sciences, and
4
School of Applied Sciences, Royal Melbourne Institute of Technology University, Bundoora, Victoria, Australia
2
Submitted 15 November 2005; accepted in final form 8 December 2005
diacylglycerol; ceramide; L6 myotube; stearoyl CoA desaturase 1
of triacylglycerol (TAG) in obesity is associated with the development of insulin resistance
and Type 2 diabetes. Skeletal muscle is a major site for
insulin-stimulated glucose disposal (4), and the accumulation
THE EXCESSIVE ACCUMULATION
* These authors contributed equally to this work.
Address for reprint requests and other correspondence: M. J. Watt, St.
Vincent’s Institute of Medical Research, 9 Princess St., Fitzroy 3065, Victoria,
Australia (e-mail: [email protected]).
http://www. jap.org
of TAG within lipid droplets in skeletal muscle is positively
correlated to the severity of insulin resistance (13, 22, 32).
However, insulin sensitivity and intramyocellular TAG
(IMTG) increase after endurance exercise training (6, 15, 18),
and TAG storage protects Chinese hamster ovary cells from
lipotoxicity (25), indicating that IMTG per se does not cause
insulin resistance but may be a proxy for other lipid metabolites that directly interfere with insulin signaling. Indeed, skeletal muscle insulin resistance is mediated by intramyocellular
accumulation of fatty acyl-CoA metabolites, such as diacylglycerols (DAG) and ceramides, which are elevated in insulinresistant states (1, 23, 36, 51) and directly interfere with insulin
signal transduction (8, 21, 41, 45, 51).
Whereas increased availability of fatty acids has been linked
to skeletal muscle insulin resistance (3, 5), it appears that the
type of fatty acid is also critical. Epidemiological evidence
suggests that the consumption of a Western diet that is high in
saturated fats closely correlates with the development of insulin resistance in humans (26, 33). Direct examination in muscle
cells in vitro indicates that saturated fatty acids cause insulin
resistance whereas unsaturated fatty acids exert a protective
effect or even improve insulin sensitivity (9, 29). Saturated fats
are less readily oxidized (14, 24) and accumulate as DAG and
ceramide in vitro, whereas mono- and polyunsaturated fats
accumulate as IMTG or free fatty acids (FFA) (14, 29, 41),
thus providing a link between fatty acid subtype and insulin
resistance. Numerous dietary studies in rodents and humans
indicate that saturated fat significantly worsens insulin resistance, whereas monounsaturated and polyunsaturated fatty acids have a less pronounced effect or even improve insulin
sensitivity (for review, see Ref. 37).
The biochemical and molecular processes linking saturated
fats to insulin resistance remain unresolved but may relate to
altered membrane phospholipid fatty acid composition and
membrane fluidity and stability (43), changes in lipogenic gene
transcription (10), the type of fatty acids within TAG (2, 28),
and direct interference with insulin signaling (8, 21, 41, 45,
51). Lipid metabolites that interfere with insulin signal transduction accumulate in tissues of insulin-resistant animals (23,
36, 40, 47, 51); however, the effects of markedly altering the
types of dietary fatty acids on the accumulation of these lipid
metabolites in whole animals are unknown. The primary aim of
the present study was to examine the role of diets rich in either
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
8750-7587/06 $8.00 Copyright © 2006 the American Physiological Society
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Lee, Jong Sam, Srijan K. Pinnamaneni, Su Ju Eo, In Ho
Cho, Jae Hwan Pyo, Chang Keun Kim, Andrew J. Sinclair,
Mark A. Febbraio, and Matthew J. Watt. Saturated, but not
n-6 polyunsaturated, fatty acids induce insulin resistance: role
of intramuscular accumulation of lipid metabolites. J Appl Physiol
100: 1467–1474, 2006. First published December 15, 2005;
doi:10.1152/japplphysiol.01438.2005.—Consumption of a Western
diet rich in saturated fats is associated with obesity and insulin
resistance. In some insulin-resistant phenotypes this is associated with
accumulation of skeletal muscle fatty acids. We examined the effects
of diets high in saturated fatty acids (Sat) or n-6 polyunsaturated fatty
acids (PUFA) on skeletal muscle fatty acid metabolite accumulation
and whole-body insulin sensitivity. Male Sprague-Dawley rats were
fed a chow diet (16% calories from fat, Con) or a diet high (53%) in
Sat or PUFA for 8 wk. Insulin sensitivity was assessed by fasting
plasma glucose and insulin and glucose tolerance via an oral glucose
tolerance test. Muscle ceramide and diacylglycerol (DAG) levels and
triacylglycerol (TAG) fatty acids were also measured. Both high-fat
diets increased plasma free fatty acid levels by 30%. Compared with
Con, Sat-fed rats were insulin resistant, whereas PUFA-treated rats
showed improved insulin sensitivity. Sat caused a 125% increase in
muscle DAG and a small increase in TAG. Although PUFA also
resulted in a small increase in DAG, the excess fatty acids were
primarily directed toward TAG storage (105% above Con). Ceramide
content was unaffected by either high-fat diet. To examine the effects
of fatty acids on cellular lipid storage and glucose uptake in vitro, rat
L6 myotubes were incubated for 5 h with saturated and polyunsaturated fatty acids. After treatment of L6 myotubes with palmitate
(C16:0), the ceramide and DAG content were increased by two- and
fivefold, respectively, concomitant with reduced insulin-stimulated
glucose uptake. In contrast, treatment of these cells with linoleate
(C18:2) did not alter DAG, ceramide levels, and glucose uptake
compared with controls (no added fatty acids). Both 16:0 and 18:2
treatments increased myotube TAG levels (C18:2 vs. C16:0, P ⬍
0.05). These results indicate that increasing dietary Sat induces insulin
resistance with concomitant increases in muscle DAG. Diets rich in
n-6 PUFA appear to prevent insulin resistance by directing fat into
TAG, rather than other lipid metabolites.
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DIETARY FATS, MUSCLE LIPIDS, AND INSULIN RESISTANCE
METHODS
Animal Experiments
Twenty-four male Sprague-Dawley rats (initial body mass 95–110
g) were obtained from Samtako, Bio Korea (Kyoung-Ki, Korea) and
housed four per cage in an environmentally controlled laboratory
(temperature 22 ⫾ 1°C and relative humidity 55 ⫾ 2%) with a
12:12-h light-dark cycle (light, 0700 to 1900). In the week before any
experimental intervention, animals were given ad libitum access to
standard rodent chow (Samtako, Bio Korea). Food intake was not
determined in this study because the polyunsaturated fatty acid diet
was too “liquid” to obtain accurate measures of the food remaining in
cages. The Animal Ethics Committee of Korea National Sport University approved all procedures performed in this study.
After 1 wk, rats were divided into one of three experimental diet
groups. Control rats (Con; n ⫽ 8) were fed standard rat chow (61.8%
carbohydrate, 15.7% fat, 22.5% protein expressed as percentage of
total caloric intake) purchased from Samtako (Bio Korea). The other
groups consumed high-fat diets (24.3% carbohydrate; 52.8% fat;
22.9% protein) that consisted primarily of saturated fatty acids (Sat;
n ⫽ 8) derived from lard and coconut oil or polyunsaturated fatty
acids (PUFA; n ⫽ 8) derived from safflower oil (ICN Biomedicals).
The main fatty acid in the PUFA diet was linoleic acid (18:2, n-6).
Animals were provided with food and water ad libitum throughout the
experimental period. Animals were maintained on their diets for 8 wk
before testing.
Oral Glucose Tolerance Test
After the 8-wk experimental period, rats were overnight fasted and
weighed and underwent an oral glucose tolerance test (OGTT).
Glucose (20% wt/vol solution) was administered by oral gavage at a
dose of 2.0 g/kg body mass, and blood was obtained from a tail vein
in unrestrained conscious rats before glucose administration and at 15,
30, 60, and 120 min thereafter. At least 2 days after OGTT and after
an overnight fast (10 h), rats were anesthetized by intraperitoneal
J Appl Physiol • VOL
injection of pentobarbital sodium (60 mg/kg body mass) and the
vastus lateralis muscle was rapidly dissected out, immediately frozen
in liquid nitrogen, and stored at ⫺80°C until analyses. A blood sample
was drawn from the femoral artery, and rats were killed by heart
removal.
Analyses
Measurement of skeletal muscle lipid metabolites. IMTG content
was analyzed as previously described (49). Freeze-dried muscle was
powdered and cleaned of all visible connective tissue and blood under
magnification. Lipid was extracted by a Folch extraction, the TAG
was saponified in an ethanol-KOH solution at 60°C, and glycerol
content was determined fluorometrically. DAG and ceramide were
extracted and quantified according to the methods of Preiss et al. (35)
with minor modifications. Briefly, lipids were extracted from freezedried, powdered muscle by using chloroform-methanol-PBS⫹0.2%
SDS (1:2:0.8 vol/vol/vol). DAG kinase and 32P-ATP (15 mCi/mmol
cold ATP) were added to extracts, and the reaction was stopped with
chloroform-methanol (2:1). Samples were spotted onto thin-layer
chromatography (TLC) plates and developed to two-thirds of the total
plate length in a solvent consisting of chloroform-acetone-methanolacetic acid-water (100:40:20:20:10). Bands corresponding to DAG
and ceramide were identified against standards after phospho-imaging, dried, scraped from the TLC plate, and counted in a liquid
scintillation analyzer (Tri-Carb 2500TR, Packard, Canberra,
Australia).
Determination of TAG fatty acid composition. Freeze-dried muscle
(⬃100 mg) was extracted with chloroform-methanol (2:1 by volume)
containing 10 mg/l of butylated hydroxytoluene. After storage of the
samples overnight at 4°C, each sample was filtered, rinsed with an
additional volume of extracting solvent, and partitioned against 0.9%
NaCl. This was evaporated under nitrogen gas, and the lipids were
reconstituted in chloroform. Samples were spotted onto TLC plates
and developed in a solvent consisting of petroleum ether-diethyl
ether-acidic acid (85:15:3 vol/vol/vol), and the TAG fraction was
identified against a standard (Nu-Chek, 18 5A) and scraped into a vial.
Methyl esters of fatty acids of the TAG extract were prepared by
transmethylation using 5% H2SO4 in methanol. The fatty acid methyl
esters were separated by capillary gas liquid chromatography using a
50 m ⫻ 0.32 mm (ID) fused silica, bonded-phase column (BPX70,
SGE, Melbourne, Australia) with helium as carrier gas at a flow rate
of 43 cm/s. The column oven was maintained at 125°C for 3 min and
increased at a rate of 8°C/min to 220°C and this temperature was
maintained for the duration of the run. Fatty acids were identified by
comparison with standard mixtures of fatty acid methyl esters and the
results are expressed as percent of the total TAG fraction.
Evaluation of mRNA content by RT-PCR. Approximately 20 mg of
muscle were extracted for total RNA by a modification of the acid
guanidium thiocyanate-phenol chloroform extraction method. The
total RNA was quantified by spectrophotometry (Hitachi U 2000
spectrophotometer), and 100 ng of each total RNA sample were
reverse transcribed. Control samples were also analyzed where all the
above reagents are added to RNA samples except the MultiScribe
reverse transcriptase. The reverse transcription reactions were performed by use a GeneAmp PCR system 2400 (Perkin-Elmer, Wellesley, MA) with conditions at 25°C for 10 min, 48°C for 30 min, and
95°C for 5 min. Two milliliters of 0.5 mmol/l EDTA (pH 8.0) were
added to each sample and stored at ⫺20°C until further analysis.
Real-time PCR was employed to quantitate rat SCD1 mRNA. Rat
probe and primers were designed (Primer Express version 1.0 Applied
Biosystems) from the rat gene sequence accessed from Gen-Bank/
EMBL (NM_139192). The sequences of the primers and probe were
as follows: forward primer, 5⬘-CAC GCC GAC CCT CAC AA-3⬘;
reverse primer, 5⬘-TCT TTG ACA GCC GGG TGT TT-3⬘; Taqman
probe, 5⬘-TTC TTC TCT CAC GTG GGT TGG CTG CTT-3⬘.
Ribosomal 18S was used as a constitutive housekeeping control
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saturated or polyunsaturated fatty acids on skeletal muscle fatty
acid metabolite accumulation and whole-body insulin sensitivity. We hypothesized that a diet high in saturated fatty acids
would induce insulin resistance and increase the contents of
skeletal muscle DAG and ceramide, whereas a diet high in
polyunsaturated fatty acids would not affect DAG and ceramide contents or impact on insulin sensitivity. To confirm the
biochemical responses to fatty acids, independent of potential
changes in metabolic or hormonal fluxes, we also incubated rat
L6 myotubes, an in vitro skeletal muscle model, with saturated
and unsaturated fatty acids and hypothesized that the in vivo
effects would persist in vitro.
Stearoyl CoA desaturase 1 (SCD1) is an endoplasmic reticulum-bound enzyme that converts saturated fatty acids (primarily 16:0; 18:0) to monounsaturated fatty acids. Global
SCD1 deficiency produces a lean, obesity-resistant, insulinsensitive phenotype (31); however, preventing the ability to
desaturate fatty acids should lead to insulin resistance because
saturated fats accumulate as DAG and ceramide. Although
polyunsaturated fatty acids reduce and saturated fatty acids
increase SCD1 expression in liver (30), the role of dietary fatty
acids on SCD1 expression and activity in skeletal muscle is
unknown, despite the importance of skeletal muscle in fuel
metabolism and insulin resistance. Accordingly, the second
aim of this study was to investigate the role of dietary fatty
acids on skeletal muscle SCD1 gene and protein expression in
vivo.
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DIETARY FATS, MUSCLE LIPIDS, AND INSULIN RESISTANCE
Cell Culture Experiments
L6 myoblasts were maintained at 37°C in 5% CO2-95% O2
humidified air in ␣ modified essential medium ⫹ 10% FBS. Differentiation was induced by switching to medium containing 2% FBS
when the myoblasts were ⬃70% confluent. Experimental treatments
were started after 2 days, by which time nearly all of the myoblasts
had fused to form myotubes. Cells were incubated with 0.5 mmol/l
palmitate (16:0) or 0.5 mmol/l linoleate (18:2) conjugated to 2% BSA,
or 2% BSA alone (Con) for 5 h, and TAG, DAG and ceramide content
were subsequently determined (described above). For determination
of 2-deoxy-D-[14C]glucose uptake, cells were grown as described and
incubated in 0.5 mM fatty acids or BSA for 5 h. Cells were washed
with warm PBS and incubated in MEM without (basal) or with 100
nmol/l insulin for 30 min. The medium was removed and 2-deoxyD-[14C]glucose uptake (0.5 ␮Ci/ml, 10 ␮mol/l cold 2-DG) was added.
The assay was stopped after 20 min by the addition of ice-cold PBS,
and cells were lysed in 0.3 M NaOH. Radioactivity was determined by
liquid scintillation counting.
Statistics
Data are expressed as means ⫾ SE. Statistical analysis were
performed by a one-way analysis of variance with a Student-Newman-Keuls post hoc test. Statistical significance was set at P ⬍ 0.05.
RESULTS
Skeletal Muscle Lipid Content Is Influenced by
Dietary Content
Lipids were assessed in skeletal muscle after an 8-h fast.
Skeletal muscle TAG was increased in Sat and PUFA compared with Con, and PUFA TAG content tended (P ⫽ 0.08) to
be greater than Sat (Fig. 2A). Skeletal muscle DAG content
was increased in Sat and PUFA compared with Con (Fig. 2B).
DAG content was greater in Sat vs. PUFA. Ceramide content
in rodent muscles was variable and was not affected by dietary
content (Fig. 2C). Dietary fatty acid composition exerted a
profound effect on the fatty acid profile in skeletal muscle
TAG. Sat increased the proportion of saturated fatty acids and
decreased unsaturated fatty acids within TAG compared with
Con (Fig. 3A). The elevation in saturated fats with Sat is
attributed to increases in 12:0, 14:0, and 18:0 (Fig. 3B).
Interestingly, 18:1 was markedly elevated and 18:2 decreased
in Sat vs. Con. PUFA increased the percentage of unsaturated
fats in TAG, which was largely due to increased 18:2. PUFA
also decreased the proportion of all saturated fatty acids in
TAG compared with Con and Sat (Fig. 3B). The proportion of
saturated fatty acids was 32, 42, and 17% for Con, Sat, and
PUFA, respectively.
SCD1 Is Increased by a Saturated Fatty Acid Diet
SCD1 mRNA was unaffected by a diet high in polyunsaturated fatty acids and tended (P ⫽ 0.11) to increase with
saturated fatty acid feeding (Fig. 4A). We examined SCD1
protein expression by Western blot and report increased SCD1
in Sat compared with Con and PUFA (Fig. 4B). An index of
SCD1 enzyme activity, which was calculated as the relative
amount of product (16:1) to the corresponding substrate (16:0),
Table 1. Fasting plasma metabolites in rats fed a chow diet
or high-fat diet consisting of either saturated or
polyunsaturated fatty acids
FFA, ␮mol/l
Glucose, mmol/l
Insulin, pmol/l
Glucose Tolerance Is Reduced in Rats Fed Diets High in
Saturated, but not n-6 Polyunsaturated, Fatty Acids
Animal weights were not different between groups after 8
wk diet (Con, 391 ⫾ 9; Sat, 409 ⫾ 7; PUFA 380 ⫾ 4 g). Both
J Appl Physiol • VOL
high-fat diets increased plasma FFA by ⬃30% but were without effect on fasting blood glucose (Table 1). Fasting plasma
insulin was increased in animals fed saturated fat compared
with chow-fed animals and reduced in animals fed polyunsaturated fat (Table 1). The homeostasis model assessment of
insulin resistance (HOMA-IR) index was increased in Sat and
decreased in PUFA compared with Con (Fig. 1A). These data
indicate that Sat rats were insulin resistant and that insulin
sensitivity was improved in PUFA rats. An OGTT was also
performed, and blood glucose and insulin levels were measured. The glucose area under the curve was not different
between groups (Fig. 1B); however, plasma insulin levels were
elevated in Sat compared with Con, indicating insulin resistance in these animals (Fig. 1B). Conversely, plasma insulin
levels were decreased in PUFA compared with Con, indicating
improved insulin sensitivity (Fig. 1C).
Con
Sat
PUFA
354⫾29
3.99⫾0.16
27⫾5
460⫾23*
4.61⫾0.15
76⫾24*
448⫾25*
4.26⫾0.10
13⫾3*†
Values are means ⫾ SE; n ⫽ 8. Con, chow diet; Sat, saturated fatty acid
high-fat diet; PUFA, polyunsaturated fatty acid high-fat diet. *Different from
Con, †different from Sat, P ⬍ 0.05.
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(Applied Biosystems, Foster City, CA). The concentrations of forward
primer, reverse primer, probe, and cDNA were 300 nM, 300 nM, 100
nM, and 10 ng, respectively. Gene expression was quantified by a
multiplex comparative critical threshold method (Bio-Rad i Cycler
IQTM, Hercules, CA).
Determination of protein content and activity. Muscle was homogenized (Polytron; Brinkman Instruments, Westbury, NY) in ice-cold
buffer containing 20 mM HEPES, 1 mM DTT, 1 mM Na4P2O7, 2 mM
EDTA, 1% Triton X-100, 10% glycerol (vol/vol), 3 mM benzamidine,
1 mM phenylmethylsulfonyl fluoride, 5 ␮l/ml phosphatase inhibitor
cocktail 2 (Sigma), and 5 ␮l/ml protease inhibitor cocktail (Sigma)
and rotated for 40 min at 4°C. Homogenates were centrifuged at
16,000 g for 45 min, and the supernatant was removed and rapidly
frozen in liquid nitrogen. Protein concentration of the muscle lysates
was subsequently determined according to the bicinchoninic acid
method (Pierce Kit, Progen Industries, Darra, QLD, Australia). Muscle lysates were solubilized in Laemmeli sample buffer and boiled for
5 min, resolved by SDS-PAGE on 12% polyacrylamide gels, transferred to a nitrocellulose membrane, blocked with 5% milk, and
immunoblotted with the primary antibody overnight. The SCD1
antibody was obtained from Santa Cruz Biotechnology (Santa Cruz,
CA). After incubation with horseradish peroxidase-conjugated secondary antibody (Amersham Biosciences, Castle Hill, NSW, Australia), the immunoreactive proteins were detected with enhanced chemiluminescence (Amersham BioSciences) and quantified by densitometry (ChemiDoc XRS, Bio-Rad Laboratories, Regents Park, NSW,
Australia).
Blood metabolite and hormone analysis. Whole blood was obtained
from the tail vein and used for blood glucose (Glucocard II, KDK)
determination before anesthesia. Whole blood was transferred to an
EDTA-administered tube and centrifuged (5,000 rpm for 15 min), and
the plasma was collected. FFA concentration was determined by an
enzymatic colorimetric method (NEFA C test kit, Wako), and insulin
concentration was measured by enzymatic immunoassay ELISA kit
(Mercodia AB, Uppsala, Sweden).
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DIETARY FATS, MUSCLE LIPIDS, AND INSULIN RESISTANCE
was increased (P ⬍ 0.05) in Sat compared with Con and
reduced (P ⬍ 0.05) in PUFA (Fig. 4D).
Effect of Palmitate and Linoleate on Fatty Acid Metabolite
Content and Glucose Uptake in L6 Myotubes
DISCUSSION
Fig. 1. Effect of dietary fatty acid composition on insulin sensitivity in rats. A:
the homeostasis model assessment of insulin resistance (HOMA-IR) index was
calculated from fasting blood glucose and insulin levels. HOMA-IR ⫽ (fasting
glucose ⫻ fasting insulin)/22.5, where glucose is expressed as mmol/l and
insulin ␮U/l. In separate experiments, overnight fasted rats were provided with
an oral glucose load and blood samples were taken for 120 min. Con, chow-fed
control; Sat, saturated fatty acid diet; PUFA, polyunsaturated fatty acid diet. B:
glucose area under the curve. C: plasma insulin were determined from tail
bleeds. Values are means ⫾ SE, n ⫽ 8. *Different from chow-fed control
(Con); †different from polyunsaturated fatty acid diet (PUFA), P ⬍ 0.05.
The present study demonstrates that a diet high in saturated
fat induces muscle DAG accumulation and saturation of the
TAG pool and is associated with whole-body insulin resistance. Conversely, animals fed a diet high in n-6 polyunsaturated fat retained insulin sensitivity despite small increases in
muscle DAG, which may result from enhanced TAG storage.
Thus fatty acid oversupply per se is not the major determinant
of fat-induced insulin resistance, but rather dietary fat composition (42, 43).
An important caveat of the present study relates to the
interpretation of insulin resistance. The OGTT and HOMA-IR
assessments do not allow for assessment of tissue-specific
Fig. 2. Effect of dietary fatty acid composition on muscle lipid metabolites. Overnight fasted rats were killed and muscle triacylglycerol (A), diacylglycerol (B),
and ceramide (C) were determined. Values are means ⫾ SE, n ⫽ 8. P ⬍ 0.05 *vs. Con, †vs. Sat.
J Appl Physiol • VOL
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To enable biochemical studies to be conducted in the absence of possible confounding factors, such as alterations in
circulating metabolites and hormones, we performed experiments in L6 myotubes. Although the metabolic action of these
cells to insulin is blunted and direct comparisons with in vivo
studies should be interpreted with caution, L6 myotubes nevertheless possess the proteins required for fatty acid and glucose uptake and metabolism. Glucose uptake was determined
in L6 myotubes after 5 h treatment with 0.5 mM palmitate
(saturated) or linoleate (polyunsaturated) fatty acids. Palmitate
decreased insulin-stimulated glucose uptake, whereas linoleate
pretreatment did not affect glucose uptake (Fig. 5A). The
addition of palmitate to culture media resulted in significant
DAG (5-fold) and ceramide (2-fold) accumulation (Fig. 5, C
and D). TAG content determined by fluorometric analysis after
5-h incubation revealed a small increase with palmitate (Fig.
5B). TAG was increased by linoleate treatment (Fig. 5B),
whereas DAG and ceramide content were unchanged (Fig. 5, C
and D). These data indicate that palmitate-induced ceramide
and DAG accumulation are associated with insulin resistance
in skeletal muscle cell culture (8, 29, 41). They also indicate
that linoleate promotes synthesis of TAG and does not affect
insulin-stimulated glucose uptake.
DIETARY FATS, MUSCLE LIPIDS, AND INSULIN RESISTANCE
1471
Fig. 3. Effect of dietary fatty acid composition on skeletal muscle triacylglycerol fatty acids. Overnight fasted rats were killed, and muscle triacylglycerols
were extracted and analyzed for fatty acid composition by gas chromatography. A: percent saturated and unsaturated fatty acids within triacylglycerols. B:
fatty acid profile within triacylglycerol. Values are means ⫾ SE, n ⫽ 8. *P ⬍
0.05, vs. Con.
effects on insulin-stimulated glucose uptake. Although skeletal
muscle accounts for ⬎80% of insulin-mediated glucose disposal (4) and is likely to represent events occurring at the
whole-body level, we cannot definitively conclude that the
observed whole-body changes are due to altered skeletal muscle function. In this regard, it appears that glucose intolerance
may only becomes apparent when insulin resistance occurs in
numerous tissues (50). Thus the present data relating to insulin
resistance should be interpreted with these considerations in
mind.
DAGs and ceramides antagonize insulin signaling in vitro,
and this is the first study to directly examine the effects of
dietary fatty acid composition on the accumulation of these
lipid metabolites in vivo. DAGs are increased in fatty acidinduced insulin resistance (19, 51) and are proposed to interfere with insulin signaling via novel PKC activation (51). In
the present study, DAG was elevated in Sat and occurred
concomitantly with insulin resistance, observations that are
consistent with the present and previous studies in muscle cell
culture that ascribe a direct role for saturated, but not unsaturated, fatty acids in this process (7, 9, 14, 29). However, our in
vivo finding that DAGs were moderately elevated in PUFA
rats, where insulin action was improved, indicates that total
DAG accumulation may not be essential for insulin resistance
and that other factors such as a critical threshold of intracellular
DAG, cellular localization of DAG, or the molecular DAG
species may be important mediators of insulin resistance.
Collectively, these data indicate that an abundance of dietary
saturated fatty acids induce insulin resistance, possibly via a
marked increase in DAG content, whereas smaller increases in
DAG were not associated with insulin resistance in animals fed
an isocaloric diet rich in polyunsaturated fatty acids.
J Appl Physiol • VOL
Fig. 4. Effect of dietary fatty acid composition on stearoyl CoA desaturase 1
(SCD1) expression. Muscle from overnight fasted rats was rapidly dissected
and examined for SCD1 mRNA (A) and SCD1 protein (B). A representative
immunoblot is shown in B. C: SCD1 activity index. Values are means ⫾ SE,
n ⫽ 8. P ⬍ 0.05, *vs. Con, †vs. PUFA.
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Ceramide is a second messenger in the sphingomyelin signaling pathway and is produced via the hydrolysis of sphingomyelin and from de novo synthesis (palmitoyl-CoA and
serine). Ceramides antagonize insulin signaling (8, 41, 45), and
ceramide accumulation correlates with the development of
skeletal muscle insulin resistance in vivo (1, 16, 44, 47). In
contrast with our hypothesis, dietary fatty acid composition did
not affect muscle ceramide content. This was unexpected
because long-chain saturated fatty acids are thought to exclusively act as the substrate for ceramide de novo synthesis (46);
however, a recent study that infused a lipid emulsion primarily
composed of linoleate (18:2) induced ceramide generation and
insulin resistance in humans (44). A dissociation between
ceramide content and insulin sensitivity has been observed
previously (19, 23, 51) and, consistent with the present findings, indicates that ceramides may not induce insulin resistance
in vivo.
IMTG content positively correlates with insulin resistance
(13, 22, 32), which has led to the assumption that excess IMTG
storage is undesirable. However, in some circumstances, such
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DIETARY FATS, MUSCLE LIPIDS, AND INSULIN RESISTANCE
Fig. 5. Lipid storage and glucose uptake in L6
myotubes after 5 h of treatment with palmitate or
linoleate. A: 2-deoxyglucose uptake with or without
insulin. B: L6 myotube triacylglycerol. C: diacylglycerol. D: ceramide content. Values represent 2
independent experiments, n ⫽ 4 per experiment.
P ⬍ 0.05, *vs. Con, †vs. PUFA.
J Appl Physiol • VOL
the lipid-lowering agent bezafibrate reduces the saturation in
muscle TAGs and improves insulin sensitivity (28). Here, we
show that a greater degree of desaturation in TAG is associated
with improved insulin sensitivity, and, conversely, an increase
in saturation correlates with insulin resistance.
SCD1 converts SATs to monounsaturated fatty acids and
impacts on metabolic function. Our current understanding of
the biological role of SCD1 derives from studies in liver and
others conducted in SCD1-null (abJ/abJ, SCD-1⫺/⫺) mice (11,
31) that demonstrate SCD1 deficiency to induce a leaner
phenotype and resistance to diet-induced obesity (11, 31).
There are very limited data describing SCD1 regulation in
skeletal muscle, a tissue that displays marked structural, functional, and metabolic differences than liver. We show that
SCD1 gene and protein expression is not different between
chow-fed and polyunsaturated fat-fed rats, which is not consistent with the findings in liver that polyunsaturated fatty acids
oppose the induction of sterol regulatory-element binding protein 1c and inhibit SCD1 gene expression (10, 48). In contrast,
SCD1 protein expression was elevated in insulin-resistant Sat
rats. Regarding the known function of SCD1, these data indicate that rather than being a causative factor in obesity and
insulin resistance, increasing skeletal muscle SCD1 may act as
a protective mechanism intended to reduce the incorporation of
saturated fatty acids into bioactive lipid species such as DAGs
and ceramides. Studies that directly test this hypothesis warrant
further investigation.
Chronic high-fat feeding induces insulin resistance in rodents; however, the role of specific dietary fats is not well
characterized. Previous reports indicate that insulin resistance
(20, 43) or glucose intolerance (27) occurs in rats fed a diet
high in n-6 PUFAs, whereas rats fed diets high in n-3 and with
low n-6 –to–n-3 ratios maintain normal insulin sensitivity. In
this study, we observed an ⬃20% improvement in HOMA-IR
in rats fed a PUFA diet consisting mainly of n-6 fatty acids
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as after endurance exercise training, increased TAG is associated with enhanced insulin sensitivity (6, 15, 18, 44). In the
present study, IMTG was increased in rats fed a Sat diet and,
to a greater extent, in rats on the PUFA diet. These data are
consistent with our studies in L6 myotubes demonstrating
marked increases in IMTG with linoleate compared with
palmitate treatment. Unsaturated fats promote TAG accumulation in various cell types (e.g., cardiac, pancreatic, Chinese
hamster ovary cells) (see Ref. 25), and this and previous
studies in skeletal muscle culture (29) have demonstrated
increased TAG storage after incubation with unsaturated fats.
Thus unsaturated fatty acids may protect cells from “lipotoxicity” by promoting uptake of fatty acids into an inert triglyceride lipid pool, rather than bioactive lipids such as DAGs and
ceramides (25). The observations in PUFA rats of elevated
muscle TAG and reduced DAG content, lower basal plasma
insulin, and enhanced insulin sensitivity compared with Sat
supports this possibility. The reason(s) for IMTG accumulation
was not addressed in this study but may relate to the preference
of DAG acyltransferase (converts DAG to TAG) for unsaturated rather than saturated fatty acids (12, 39).
The fatty acid profile of different body tissues at least
partially reflects the fatty acid composition of the diet. In this
study we compared the fatty acid profile of TAG between
groups. The 8-wk Sat diet increased the degree of saturation
within skeletal muscle TAG, which was due to increases in
short (12:0, 14:0)- and long (18:0)-chain fatty acids. Whether
an increase in TAG saturation contributes to insulin resistance
in unknown. It is possible that the excess stearate (18:0) within
this pool is hydrolyzed and provides a substrate for DAG and
ceramide production, thereby affecting insulin signaling. In
contrast with Sat, PUFA increased the degree of unsaturation
within IMTG, which was primarily due to increased linoleate
(18:2). Increased unsaturation of the muscle membrane fatty
acids is associated with improved insulin sensitivity (43), and
DIETARY FATS, MUSCLE LIPIDS, AND INSULIN RESISTANCE
ACKNOWLEDGMENTS
Present address for J. S. Lee: Department of Physical Education, Faculty of
Physical Education and Sport Leisure Studies, Daegu University, 15 Naeri,
Jinryang, Gyeongsan, Gyeongbuk, 712-714, South Korea.
GRANTS
This work was supported by a grant from the Australian Research Council
(M. A. Febbraio, M. J. Watt, DP0450436) and Royal Melbourne Institute of
Technology University (Virtual Research Institute in Nutrition scheme). M. J.
Watt is supported by a Peter Doherty postdoctoral fellowship, and M. A.
Febbraio a senior research fellowship from the National Health and Medical
Research Council of Australia. J. S. Lee was supported by Korea Research
Foundation Grants KRF-2002-037-G00028 and KRF-2003-037G00067.
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