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DIABETES, VOL. 50, JANUARY 2001 123
Prolonged Inhibition of Muscle Carnitine
Palmitoyltransferase-1 Promotes Intramyocellular
Lipid Accumulation and Insulin Resistance in Rats
Robert L. Dobbins, Lidia S. Szczepaniak, Brandon Bentley, Victoria Esser, Jeffrey Myhill,
and J. Denis McGarry
Cross-sectional studies in human subjects have used
1
H magnetic resonance spectroscopy (HMRS) to demon-
strate that insulin resistance correlates more tightly
with the intramyocellular lipid (IMCL) concentration
than with any other identified risk factor. To further
explore the interaction between these two elements in
the rat, we used two strategies to promote the storage
of lipids in skeletal muscle and then evaluated subse-
quent changes in insulin-mediated glucose disposal. Nor-
mal rats received either a low-fat or a high-fat diet
(20% lard oil) for 4 weeks. Two additional groups (low-
fat + etoxomir and lard + etoxomir) consumed diets con-
taining 0.01% of the carnitine palmitoyltransferase-1
inhibitor, R-etomoxir, which produced chronic blockade
of enzyme activity in liver and skeletal muscle. Both the
high-fat diet and drug treatment significantly impaired
insulin sensitivity, as measured with the hyperinsulinemic-
euglycemic clamp. Insulin-mediated glucose disposal
(IMGD) fell from 12.57 ± 0.72 in the low-fat group to
9.79 ± 0.59, 8.96 ± 0.38, and 7.32 ± 0.28 µmol · min
–1
· 100 g
–1
in the low-fat + etoxomir, lard, and lard + etoxomir
groups, respectively. We used HMRS, which distinguishes
between fat within the myocytes and fat associated with
contaminating adipocytes located in the muscle bed, to
assess the IMCL content of isolated soleus muscle. A
tight inverse relationship was found between IMGD
and IMCL, the correlation (R = 0.96) being much stronger
than that seen between IMGD and either fat mass or
weight. In conclusion, either a diet rich in saturated fat
or prolonged inhibition of fatty acid oxidation impairs
IMGD in rats via a mechanism related to the accumu-
lation of IMCL. Diabetes 50:123–130, 2001
E
vidence indicates that abnormalities of lipid metab-
olism can negatively impact insulin-mediated
glucose disposal (IMGD) in skeletal muscle. The
elevated skeletal muscle lipid stores found in many
models of insulin resistance are consistent with this idea
(1–10). For example, muscle triglyceride levels (as measured
in biopsy samples) are increased in patients with type 2 dia-
betes (11) and, in nondiabetic individuals, appear to correlate
with insulin resistance better than measures of adiposity,
such as BMI or percent body fat (9). Because the measure-
ment of total triglycerides in tissue extracts cannot discrim-
inate between fat within the myocyte and fat associated
with surrounding adipose tissue, we have used
1
H magnetic
resonance spectroscopy (HMRS) to provide a noninvasive
measure of the lipid within the muscle cells (12,13). Distin-
guishing intramyocellular lipid (IMCL) from extramyocellular
lipid (EMCL) concentrations provides a quantitative mea-
sure of true cell-associated lipids that can only be qualitatively
assessed in muscle biopsy samples by oil-red O staining or
electron microscopy. It also greatly reduces the variability
inherent in the measurement of cell-associated lipids by use
of conventional chemical analyses of biopsy samples (13,14)
In cross-sectional studies with humans, we found that
insulin resistance correlates more tightly with IMCL than with
any other factor, including BMI, percent body fat, waist-to-hip
ratio, or age (12). Other groups have recently reported simi-
lar findings (15–17). Furthermore, an acute intervention,
such as infusion of a lipid emulsion to augment tissue uptake
of fatty acids in normal humans, antagonizes insulin-stimulated
uptake and phosphorylation of glucose in muscle (18,19).
The same is true in rat muscle previously exposed to elevated
free fatty acid (FFA) levels (20). Importantly, more prolonged
exposure of rats to a diet containing large quantities of fat elic-
its insulin resistance that is associated with the accumulation
of fatty acyl-CoA (8) and triglycerides (1,10) in skeletal mus-
cle. Because rodents receiving high-fat diets have an increased
adipose tissue mass, it is evident that variability in the amount
of adipose tissue within the muscle bed could bias measure-
ments of muscle triglycerides made from extracts of whole
muscle (1,10). We deemed it important, therefore, to verify
that high-fat diets elicit insulin resistance in association with
the accumulation of IMCL when it is distinguished from adi-
pose tissue by HMRS.
Although excessive dietary fat provides a strong impetus
for the accumulation of lipids in muscle and other tissues,
the same effect is theoretically achievable by pharmaco-
logical blockade of fatty acid oxidation with the R-isomer of
ethyl-2-[6-(4-chlorophenoxy)hexyl]-oxirane-2-carboxylate
(etomoxir). After conversion to its CoA ester, R-etomoxir
covalently inhibits carnitine palmitoyltransferase (CPT)-1
and blocks the entry of fatty acids into mitochondria (21,22).
The racemic mixture of R- and S-etomoxir has hypoglycemic
effects in humans and animal models with diabetes and has
been proposed as a potentially useful therapeutic agent
From the Departments of Internal Medicine (R.L.D., L.S.S., B.B., V.E., J.M.,
J.D.M.) and Biochemistry (J.D.M.), University of Texas Southwestern Med-
ical Center at Dallas, Dallas, Texas.
Address correspondence and reprint requests to Robert L. Dobbins,
MD, PhD, University of Texas Southwestern Medical Center, Dallas, TX
75235-9135. E-mail: rdobbi@mednet.swmed.edu.
Received for publication 27 March 2000 and accepted in revised form
26 September 2000.
CPT, carnitine palmitoyltransferase; EMCL, extramyocellular lipid; FFA,
free fatty acid; HMRS,
1
H magnetic resonance spectroscopy; IMCL, intramy-
ocellular lipid; IMGD, insulin-mediated glucose disposal.
(23–25). Because prolonged treatment with high doses of
etomoxir promotes triglyceride accumulation within tissues
(as determined by chemical analysis of biopsy samples [26]),
a second objective of the present study was to determine what
effect such treatment might have on both whole-body insulin
sensitivity and IMCL in rats.
RESEARCH DESIGN AND METHODS
Animals. Male Sprague-Dawley rats weighing 125–150 g were placed in
metabolic cages at 22°C and maintained on a 12-h light-dark cycle (lights on from
10:00
A.M. to 10:00 P.M.) with ad libitum access to water and specially prepared
diets (Harlan Teklad, Madison, WI). To investigate whether a high-fat diet could
alter insulin sensitivity in association with changes in IMCL content, all animals
received either a high-fat diet enriched with highly saturated lard oil or a low-fat
diet containing only essential fatty acids for 4 weeks. The low-fat diet (TD94128)
contained primarily cornstarch and had a caloric density of 3.5 kcal/g with a
distribution of 4% fat, 75% carbohydrate, and 21% protein. The lard diet
(TD96001) had a caloric density of 4.4 kcal/g and contained 41% fat, 40% carbo-
hydrate, and 19% protein. To investigate the impact of etomoxir on insulin sen-
sitivity and IMCL, two separate groups of animals (low-fat + etoxomir and lard +
etoxomir) received diets supplemented with 0.01% R-etomoxir. Food intake was
monitored daily, and body weights were recorded twice a week.
Isolation of mitochondria and assay of CPT-1 activity. To verify sup-
pression of CPT-1 activity by etomoxir, rats in both the fed and 18 h–fasted
states were killed, and mitochondria were isolated from the liver and gas-
trocnemius muscle by method A of McGarry et al. (27). They were suspended
in 150 mmol/l KCl, 5 mmol/l Tris-HCl, pH 7.2, and CPT-1 /CPT-2 activity was
assayed as described previously in the direction palmitoyl-CoA +
L-[
14
C]car-
nitine → palmitoyl-[
14
C]carnitine + CoASH (28). Enzyme activity was nor-
malized for mitochondrial protein content (29).
Hyperinsulinemic-euglycemic clamps. Rats were anesthetized with keta-
mine (55 mg/kg) and xylazine (10 mg/kg) and fitted with carotid artery and
jugular venous catheters after 3 weeks of diet treatment (30). They were
allowed 6–8 days to recover before experiments were performed. Animals were
fasted starting at 4:00
P.M. and were studied in the conscious unrestrained
state at ~10:00
A.M. the following day. Catheters were flushed with 25 U/ml
heparinized saline (Elkins-Sinn, Cherry Hill, NJ), and the venous line was
connected for infusion of 3-[
3
H]glucose, insulin, and replacement blood
(1.5 ml/h). Replacement blood was obtained via cardiac puncture from a lit-
termate of the study animal (31). The arterial line was used for blood sampling
and was kept patent by the infusion of heparinized saline. Studies lasted
210 min and were conducted according to a modification of the procedure out-
lined by Rossetti and colleagues (31–33). A 3-µCi bolus of 3-[
3
H]glucose was
given to initiate the experiment, followed by infusion of 0.25 µCi/min for 90 min.
From 60–90 min, blood was sampled at 5-min intervals for determination of
plasma [
3
H]glucose–specific activity and content of
3
H
2
O after precipitation
with BaOH and ZnSO
4
. The tracer infusion was discontinued after 90 min and
insulin was infused at 60 mU/kg body wt for 2 min before being decreased to
3 mU
· kg
–1
· min
–1
. Plasma glucose concentrations were measured frequently,
and 25% glucose was infused to maintain a constant level of ~6 mmol/l.
Thirty minutes after the insulin was started, the 3-[
3
H]glucose tracer infusion
was bolused again and continued at 0.25 µCi/min. From 180–210 min, blood
was drawn at 5-min intervals for determination of plasma tritiated glu-
cose–specific activity and content of tritiated water. Additional samples were
obtained at 75, 90, 150, 180, and 210 min for assay of plasma FFA and triglyc-
eride levels. Remaining plasma from the 60- to 90-min and 180- to 210-min sam-
ples was pooled and frozen for subsequent assays of basal and steady-state
plasma insulin levels.
Body composition. After 4 weeks of diet treatment,
1
H spectra of the entire ani-
mal (Fig. 1A) were collected using a 4.7T Omega system with a single pulse
sequence and the following parameters: repetition time of 5 s, spectral width of
3 kHz, and 2,048 data points (34–37). Water and fat resonances were quantified
after line-fitting with NUTS software (Acorn NMR, Fremont, CA). Fat mass
and fat-free mass were computed from the ratio of fat and water signal areas
and known ratios of proton densities of fatty acid chains and water, assuming
that water comprised 72% of fat-free mass (38). This technique was validated by
previous investigators; the correlation coefficient between total body lipid and
water content measured by HMRS and classical carcass analysis was 0.97 (34).
IMCL. Overnight-fasted rats were killed for excision of the liver and bilateral
soleus muscles. All tissues were immediately frozen in liquid N
2
except for the
right soleus muscle. It was placed in saline so that a small slice (15–20 mg) could
be excised, stripped of all visible fat under 10 magnification and placed in a
5-mm NMR tube filled with deuterated saline. High-resolution proton spectra
were obtained at 37°C using a 300-MHz Varian-INOVA system and were
processed to estimate total lipid, IMCL, and EMCL content (13) by comparing
the methylene signal intensity to that of a 2-µmol formic acid standard corrected
for T
1
relaxation (39). Triglycerides from the liver and contralateral soleus mus-
cle were extracted by the method of Folch et al. (40). Aliquots of the liver extract
were saved for the measurement of total tissue triglycerides by colorimetric
assay (41), whereas the soleus muscle was fractionated using silica Sep-Pak
columns (Waters, Milford, MA) (42) for analysis of fatty acid composition by
gas-liquid chromatography (43). The average molecular weight and number of
methylene protons in the muscle triglyceride pool was used for converting pro-
ton signal intensities into milligrams of lipids.
Figure 1B provides an example of the high-resolution spectra and highlights
the two components of the methylene peak designated EMCL and IMCL.
Despite the great care taken in preparing muscle slices, spectroscopy detected
EMCL in some samples. Such interfascial adipose tissue is inevitably included
in analyses of muscle triglyceride obtained from tissue extracts (1,10), but it
can be distinguished from true IMCL when proton spectra are obtained in intact
muscle tissue (13,44–46). A detailed discussion of theoretical principles and the
practical application of HMRS for quantitative assessment of total tissue
lipids and IMCLs is beyond the scope of the current article and is provided else-
where (44–46).
The validity of the method we used in the current studies was assessed in
soleus muscle biopsies obtained from six animals consuming a standard 4%
fat diet. The HMRS measurement was completed 25–30 min after the muscle
had been excised, and the tissue was subsequently frozen at –70°C until the
neutral lipids were extracted with chloroform:methanol for biochemical mea-
surement of the total muscle triglyceride content. For comparison, an adjacent
section was immediately frozen and stored for biochemical determina-
tions. The correlation between the total muscle lipid value obtained by spec-
troscopy and the triglyceride content assayed biochemically is shown in
Fig. 2. A strong linear relationship existed between the two independent tech-
niques (R = 0.99) and the absolute difference for any value ranged between 0.23
and 1.05 mg/g tissue. The concordance between spectroscopy and biochemical
assay of a single sample was much stronger than that between biochemical
determinations of triglyceride for two separate samples taken from a single
muscle (data not shown). Figure 2 also highlights the advantage of collecting
spectra from an intact muscle preparation to differentiate between triglyceride
contained in adipocytes and myocytes. In most cases, adipose tissue
contributed only a small portion of the total lipids in the specimen, but the
contribution varied quite markedly.
Analytical procedures. Perfusate glucose concentrations were measured by
the glucose oxidase method on a Beckman Glucose Analyzer II machine.
Plasma FFA levels were determined by colorimetric assay (catalog no. 1381375;
Boehringer Mannheim, Indianapolis, IN). Plasma triglyceride concentrations and
tissue triglyceride content were measured using a colorimetric assay that was
124 DIABETES, VOL. 50, JANUARY 2001
MUSCLE LIPID AND INSULIN RESISTANCE
FIG. 1. A: Whole-body proton magnetic resonance spectra (HMRS)
showing fat and water resonances from rats receiving low-fat and
lard diets. B: HMRS of individual muscles taken from rats receiving
low-fat and lard diets. The labeled peaks represent the resonances of
protons from trimethylammonium (TMA) compounds, creatine (Cr),
and the EMCL or IMCL components of the methylene peak.
corrected for free glycerol contamination (procedure no. 337; Sigma, St. Louis,
MO). Plasma and perfusate insulin concentrations were assayed with a double-
antibody radioimmunoassay kit obtained from Linco (St. Charles, MO).
Materials. The sodium salts of R- and S-etomoxir were kindly provided by
Novo Nordisk Pharmaceuticals (Copenhagen). Radiolabeled 3-[
3
H]glucose
was obtained from NEN Life Science Products (Boston, MA) and L-[
14
C]car-
nitine was purchased from American Radiolabeled Chemical (St. Louis, MO).
Statistical analysis. Experimental results were analyzed for effects of the
diet and the drug using two-way multiple analysis of variance. The association
between IMGD and other measured parameters was assessed by linear regres-
sion analysis. All calculations were made with SigmaStat software for Windows
(SPSS, Chicago).
RESULTS
Food intake and body weight. Food intake was similar in all
diet groups, although there was a trend toward a decline in the
rats receiving R-etomoxir that did not reach statistical signifi-
cance. The average daily caloric intake was 69.6 ± 1.9, 65.2 ± 1.9,
69.7 ± 1.9, and 66.5 ± 1.0 kcal/day for animals on the low-fat,
low-fat + etoximir, lard, and lard + etoxomir diets, respec-
tively. Weight gain was similar in all cases, so that after 4
weeks, the average weights of the animals in the 4 groups
were 276 ± 5, 292 ± 7, 288 ± 9, and 270 ± 5 g, respectively.
CPT-1 activity. In animals receiving the low-fat and high-fat
diets for 4 weeks, mitochondrial CPT-1 activity in the fed
state was similar in both skeletal muscle (12.1 ± 0.1 vs. 11.8 ±
1.8 nmol · min
–1
· mg protein
–1
, respectively) and liver (8.7 ± 0.7
vs. 8.3 ± 0.2 nmol · min
–1
· mg protein
–1
). When 0.01% R-etomoxir
was added to the diets, CPT-1 activity in both fed and fasted
animals was suppressed by >95% in liver and 80–90% in skele-
tal muscle. CPT-2 was not inhibited in either tissue. Compa-
rable results were obtained after 10 and 20 days of etomoxir
treatment, indicating that the inhibitor effectively suppressed
mitochondrial fatty acid oxidation in both liver and skeletal
muscle over the time frame of these experiments.
Hyperinsulinemic-euglycemic clamps. Studies were under-
taken to determine the insulin sensitivity of the liver and skele-
tal muscle of rats that had consumed low-fat and high-fat diets
with or without etomoxir. Table 1 lists the concentrations of
plasma glucose, insulin, FFA, and triglycerides after an overnight
fast and at the end of the insulin infusion period. The fasting
plasma glucose concentration was reduced in animals receiv-
ing etomoxir, which was consistent with diminished glucose
production (Fig. 3C) via gluconeogenesis (23–25,47–50) because
glycogenolysisis in rats is completely suppressed after an
18-h fast. Exogenous insulin infusion elicited a similar increase
in plasma insulin levels in all of the groups, and plasma glucose
concentrations were maintained at euglycemic levels. Acute
administration of oxirane CPT-1 inhibitors has been known to
augment plasma triglyceride levels (50). However, we saw no
changes in fasting plasma triglyceride concentrations after
prolonged CPT-1 inhibition, although fasting plasma FFA lev-
els fell significantly.
Figure 3 illustrates the marked alterations in glucose kinet-
ics resulting from the consumption of a high-fat diet and treat-
ment with etomoxir. The glucose infusion rate during the
hyperinsulinemic period provided an index of whole-body
insulin sensitivity. The value of 13.33 ± 1.34 µmol · min
–1
· 100 g
–1
in the low-fat animals was much greater than that seen in any
DIABETES, VOL. 50, JANUARY 2001 125
R.L. DOBBINS AND ASSOCIATES
FIG. 2. Comparison of total muscle lipids () and IMCLs (), as deter-
mined by HMRS of intact muscle tissue, with total muscle triglyceride
(TG) content assayed chemically after chloroform:methanol extraction.
Soleus muscle samples from six overnight-fasted rats that had received
a 4% fat diet were analyzed.
TABLE 1
Influence of dietary fat content and R-etomoxir on plasma parameters before and during hyperinsulinemic-euglycemic clamps
Low-fat Low-fat + etomoxir Lard Lard + etomoxir
n 76 66
Glucose (mmol/l)
Basal 5.98 ± 0.09 5.28 ± 0.15* 6.18 ± 0.26 5.87 ± 0.10*
Clamp 6.18 ± 0.13 5.85 ± 0.10 6.26 ± 0.06 6.15 ± 0.14
Insulin (pmol/l)
Basal 25 ± 5 20 ± 5 40 ± 10 15 ± 5
Clamp 320 ± 30 330 ± 10 335 ± 15 300 ± 10
FFA (mmol/l)
Basal 0.68 ± 0.04 0.55 ± 0.09* 0.65 ± 0.04 0.50 ± 0.02*
Clamp 0.22 ± 0.04 0.31 ± 0.08 0.35 ± 0.04 0.30 ± 0.04
Triglycerides (mg/dl)
Basal 9.3 ± 2.2 6.0 ± 2.8 8.5 ± 1.4 12.2 ± 2.2
Clamp 9.1 ± 2.4 5.3 ± 3.4 8.4 ± 1.1 12.4 ± 3.1
Data are means ± SE. *P < 0.05 vs. rats not receiving etomoxir.
other group (Fig. 3A). Both IMGD (Fig. 3B) and insulin-
induced suppression of endogenous glucose production
(Fig. 3C) were impaired in the lard group. Glucose utilization
was 8.96 ± 0.38 µmol · min
–1
· 100 g
–1
, and glucose production
declined to only 1.61 ± 0.73 µmol · min
–1
· 100 g
–1
. For com-
parison, glucose utilization in the low-fat rats was 12.57 ±
0.72 µmol · min
–1
· 100 g
–1
, whereas production was completely
suppressed. Adding R-etomoxir to the diets impaired insulin
sensitivity regardless of dietary fat intake. Glucose was
infused at a rate of 8.19 ± 1.53 µmol · min
–1
· 100 g
–1
in the low-
fat + etoxomir group and 4.23 ± 0.67 µmol · min
–1
· 100 g
–1
in
the lard + etoxomir group. The overall reduced insulin sensi-
tivity imparted by etomoxir clearly reflected a reduction of
glucose utilization coupled with a trend toward impaired sup-
pression of endogenous glucose production. The ability of
R-etomoxir to alter glucose kinetics was specific for inhibition
of CPT-1 activity, because no changes were evident when we
supplemented the lard diet with S-etomoxir, the form lacking
the ability to block CPT-1 (data not shown).
Measures of whole-body fat and tissue lipid content. The
foregoing data clearly indicate that altering lipid dynamics,
either by increasing the dietary intake of saturated fat or
by inhibiting the clearance of lipids via the mitochondrial
-oxidation pathway, resulted in insulin resistance. There-
fore, additional experiments were undertaken to characterize
exactly what effects the various diet treatments had upon
whole-body fat mass, muscle lipid content, and hepatic triglyc-
erides. Values for these parameters are listed in Table 2. Fat
mass estimated utilizing proton magnetic resonance spec-
troscopy was reduced in the low-fat rats, whereas the other
three groups displayed similar degrees of adiposity. It was not
surprising that rats consuming the lard diet gained signifi-
cantly more fat than their counterparts in the low-fat group, but
the impact of CPT-1 inhibition on adiposity was more difficult
to interpret. The addition of etomoxir to the low-fat diet signi-
ficantly augmented body-fat content; this effect was not appar-
ent in rats consuming the high-fat diet. It has been suggested
that a defect in muscle lipid oxidation would favor storage of
lipids in adipose tissue and initiate a cascade of events culmi-
nating in obesity and insulin resistance (2,3,51). The development
of obesity in the low-fat + etoxomir group fits this sce-
nario, but it would seem that other regulatory mechanisms such
as marked insulin resistance or subtle changes in food intake
prevented additional adiposity in the lard + etoxomir group.
In keeping with previous studies with human subjects
(13,15–17), we used HMRS to quantify the lipid contained in
the soleus muscle. This muscle is primarily composed of
slow-twitch oxidative fibers (type I) that have increased
insulin sensitivity and lipid content compared with other
muscle fiber types (16,17). Table 2 reveals that the amount of
lipid measured within the muscle increased when either lard
oil or etomoxir was added to the diet. The total lipid content
of soleus muscle excised from rats in the fasted state was
significantly augmented by both dietary fat intake and CPT-1
inhibition. The unique information revealed by HMRS was that
the elevated muscle lipid stores resulted from increases in
both the extramyocellular and intramyocellular compart-
ments. Whereas no EMCLs were detected in the animals
receiving the low-fat diet, the more obese animals in the
126 DIABETES, VOL. 50, JANUARY 2001
MUSCLE LIPID AND INSULIN RESISTANCE
TABLE 2
Influence of dietary fat content and R-etomoxir on fat mass, muscle lipid content, and hepatic triglyceride content
Low-fat Low-fat + etomoxir Lard Lard + etomoxir
Fat-mass (g) 12.1 ± 0.7 23.3 ± 4.3 29.9 ± 4.5 21.2 ± 3.0
Total muscle lipid (mg/g wet wt) 3.09 ± 0.37 4.98 ± 0.36† 5.91 ± 1.42* 6.19 ± 1.48*†
Extramyocellular lipid (mg/g wet wt) 0.00 ± 0.00 1.36 ± 0.14 1.83 ± 0.97 1.23 ± 0.63
Intramyocellular lipid (mg/g wet wt) 3.09 ± 0.37 3.68 ± 0.37† 4.09 ± 0.86* 4.91 ± 1.06*†
Hepatic triglyceride (mg/g wet wt) 10.6 ± 2.2 22.6 ± 3.4† 13.6 ± 1.0* 56.9 ± 18.0*†
Data are means ± SE for four to eight determinations. *P < 0.05 vs. rats receiving the low-fat diets. †P < 0.05 vs. rats not receiving
etomoxir.
FIG. 3. Influence of dietary fat content and etomoxir (Eto) on the rates
of glucose infusion (A), glucose disposal (B), and endogenous glucose
production (C) in overnight-fasted rats before and during insulin infu-
sion at 3 mU · kg
–1
· min
–1
. Each value represents the mean ± SE for six
to seven determinations. *P < 0.05 between linked groups.
other diet groups predictably exhibited greater muscle adi-
posity. After adjusting for artifact from adipose tissue, the
IMCLs in muscle samples excised from rats were again pro-
foundly influenced by dietary fat intake and CPT-1 inhibi-
tion. Figure 4A illustrates the steep inverse relationship that
exists between IMGD, a commonly utilized index of muscle
insulin sensitivity, and total muscle lipid content. EMCL con-
tent was not independently correlated with deteriorating
insulin resistance, and it appeared to play a marginal role in
the link between IMGD and total muscle lipid content. On the
other hand, it is evident that the lipid actually contained
within muscle cells played a more pivotal role in determining
the association with IMGD (Fig. 4C). The apparent associa-
tion between IMGD and whole-body fat mass shown in Fig. 5
did not quite reach statistical significance because of the lim-
ited number of data points. These data are entirely consistent
with results of clinical studies showing that insulin resis-
tance correlates more tightly with muscle lipid content than
with percent body fat or BMI (9,12,15,52).
Hepatic triglyceride content was only measured from chlo-
roform:methanol extracts because all of the fat in the liver is con-
tained within hepatocytes (13). Inhibition of CPT-1 resulted in
a marked elevation of hepatic triglycerides exceeding the level
with increased dietary fat (Table 2). Thus, prolonged CPT-1
blockade caused fat to accumulate in the liver without any
increase in plasma FFA or triglyceride concentrations.
DISCUSSION
There is growing evidence that excessive accumulation of fat
in skeletal muscle and other tissues is an important factor in
the genesis of whole-body insulin resistance, a hallmark feature
of obesity/type 2 diabetes syndromes. Although insulin sensi-
tivity, defined in terms of glucose production and disposal
rates, can be accurately quantified using the hyperglycemic-
euglycemic clamp procedure, skeletal muscle triglyceride con-
tent has traditionally been chemically analyzed on the basis of
chloroform:methanol extraction of total lipids from biopsy
samples. This approach does not distinguish between IMCLs
and EMCLs, both of which may vary independently, particularly
in a skeletal muscle sample in which infiltration by adipocytes
may be extensive. Recently, we and others (13) have over-
come this limitation with the use of in vivo proton magnetic res-
onance spectroscopy, a procedure that readily discriminates
between IMCL and EMCL within the muscle bed. After apply-
ing this technique noninvasively in a cross-sectional study of
healthy volunteers, the degree of insulin resistance was found
to bear a tight relationship to the amount of IMCLs (12,15–17).
Because cross-sectional studies cannot truly establish a cause-
effect relationship between IMCL accumulation and insulin
resistance, we designed separate interventions to answer two
questions in the current work. First, is the insulin resistance that
is known to follow high-fat feeding in rats accompanied by ele-
vation of IMCL stores, as measured by HMRS? Second, if the
IMCL pool can be expanded by pharmacological blockade of
mitochondrial fatty acid oxidation, can insulin sensitivity also
be lowered? Both of these questions, neither of which has
been addressed as of yet, can now be answered in the affir-
mative, as discussed below.
Sequential measurement of the total triglyceride content of
a single muscle sample, first using HMRS and then by con-
ventional means following chemical extraction showed
strong agreement. However, as demonstrated with humans in
vivo (13,44,45), the primary advantage of HMRS proved to be
its ability to quantify both IMCLs and EMCLs. Thus, it was pos-
sible to show that the EMCL component represented a sub-
stantial fraction of total lipid content, even when great efforts
were made to strip all visible fat from samples. As a result, the
triglyceride level measured from the tissue extract didn’t cor-
relate as well with IMCL as it did with the sum of IMCL and
EMCL. We believe that the variability of the EMCL fraction
likely contributes significantly to the scatter in biochemi-
cally determined triglyceride levels found in different samples
taken from a single muscle (14).
Provision of a high-fat diet caused a twofold increase in
whole-body fat mass and a notable elevation of adipose tissue
in the muscle bed. Inhibition of CPT-1 with R-etomoxir also
doubled adiposity in the low-fat animals but did not further
increase fat mass in the high-fat group. Similarly, R-etomoxir
significantly increased the EMCL component of soleus mus-
cle in the low-fat but not the lard-fed rats. As predicted, an
increase in dietary fat intake elicited a striking reduction
(45%) in whole-body insulin sensitivity that was reflected in
impairment of both IMGD and suppression of endogenous
(mainly hepatic) glucose production during the hyperinsu-
linemic-euglycemic clamp. Importantly, CPT-1 inhibition neg-
atively impacted both parameters, regardless of diet.
DIABETES, VOL. 50, JANUARY 2001 127
R.L. DOBBINS AND ASSOCIATES
FIG. 4. Correlation between IMGD and soleus muscle lipid content in
rats receiving low-fat and lard diets either alone or supplemented with
the CPT-1 inhibitor, R-etomoxir (Eto). A, B, and C represent total lipid,
EMCL, and IMCL contents, respectively. Each value represents the
mean ± SE for four to seven determinations.
Fasting gluconeogenesis was modestly reduced after inhi-
bition of CPT-1. Limiting the oxidation of fatty acids during
a fast limits gluconeogenesis by depleting NADHs and ATPs
that are required for key steps of the gluconeogenic pathway
and by reducing the level of acetyl-CoA, which serves as an
allosteric activator of pyruvate carboxylase. During the hyper-
insulinemic period, glucose production was paradoxically
increased after prolonged inhibition of CPT-1. High concen-
trations of insulin normally suppress gluconeogenesis via
direct effects on the liver and an indirect mechanism linked
to the inhibition of lipolysis and a fall in hepatic fatty acid oxi-
dation (53). Because mitochondrial fatty acid oxidation was
already blocked in animals treated with R-etomoxir, we
assume that some other factor must have offset the direct
effects of insulin on hepatic gluconeogenesis in this setting.
It is known that etomoxir treatment causes triglyceride and
long-chain fatty acids to accumulate in hepatocytes, where
they activate peroxisome proliferator–activated receptor
and enhance the expression of target genes involved in per-
oxisomal fatty acid oxidation (54). Accumulated lipids might
also regulate the expression of factors participating in insulin
signaling or key enzymes of gluconeogenesis, such as glucose-
6-phosphatase, fructose 1,6-bisphosphatase, or phosphoenol-
pyruvate carboxykinase. Clearly, additional work is needed
before the complex interaction between CPT-1 inhibition
and gluconeogenesis can be fully understood.
What emerges from the present study is that the impairment
in IMGD after high-fat feeding or CPT-1 blockade correlated
strongly and inversely with the IMCL content of the soleus
muscle. We suspect that the same was true for other muscles,
although this hypothesis was not examined. It has been dif-
ficult to decipher whether the buildup of IMCL either leads
to or results from insulin resistance using cross-sectional
studies, because subjects with the highest lipid content likely
had a genetic predisposition favoring the development of
insulin resistance. In the current work, IMCLs were varied in
a group of genetically homogeneous rats. The results favor a
model in which an initial defect of lipid oxidation followed by
the accumulation of muscle lipids, particularly the intramy-
ocellular component, can lead to insulin resistance.
It seems doubtful that muscle triglycerides interfere with
insulin action in the muscle cells. Rather, we favor the view that
the triglyceride level serves as a surrogate marker for some
other fatty acid–derived entity that acts to impair insulin sig-
naling. Long-chain fatty acyl-CoA is an attractive candidate
because its intracellular concentration will invariably increase
with fat loading or after inhibition of CPT-1 (8). Long-chain fatty
acyl-CoA, perhaps through its conversion into diaclyglycerol,
might alter the activity of one or more forms of protein kinase
C with resultant impairment in insulin receptor substrate-
1–mediated activation of phosphatidylinositol 3-kinase and,
thus, GLUT4 translocation to the cell membrane (19,55–57). It is
also possible that a high concentration of one or more fatty
acyl-CoA species alters transcription/translation or acylation
of a protein(s) involved in the insulin signaling cascade. The
paradox that trained athletes are highly insulin-sensitive
despite having elevated muscle triglyceride content (58) pro-
vides further evidence that muscle triglyceride cannot be the
sole determinant of insulin sensitivity. However, lipid deposi-
tion resulting from extensive physical training could have dif-
ferent metabolic consequences than those associated with
inhibition of mitochondrial lipid oxidation. Physical training
increases the proportion of type I muscle fibers in the stressed
muscle, and these fibers generally have greater lipid content,
higher oxidative capacity, and enhanced insulin-stimulated
glucose transport relative to type 2b fibers (59,60). Prolonged
aerobic exercise depletes endogenous triglyceride stores
(45,61), and cyclical depletion and repletion of lipids in skele-
tal muscle with a high capacity for lipid oxidation may protect
against the adverse effects of muscle lipid accumulation (58).
Lipid deposition in the current model is associated with a
diminished activity of CPT-1 in skeletal muscle similar to that
recognized in human obesity (62). In a setting in which lipid
metabolism in skeletal muscle is skewed toward storage rather
than oxidation, any maneuver capable of lowering the IMCL
content should promote an increase in insulin sensitivity.
Accordingly, it is tempting to speculate that the salutary effect
of an intervention, such as the reduction of dietary fat intake
on whole-body insulin sensitivity, operates (at least in part) by
limiting lipid accumulation in muscle.
Finally, it should be emphasized that in these studies we pur-
posely chose a high dose of etomoxir to assure a major degree
of CPT-1 suppression in skeletal muscle as well as liver. Because
the muscle isoform of CPT-1 is far less sensitive to inhibition
by etomoxir-CoA than the liver isoform (21), it is possible,
using much lower doses of etomoxir, to arrange conditions so
that liver but not skeletal muscle CPT-1 is significantly inhib-
ited (unpublished observations). Accordingly, the current find-
ings do not necessarily contraindicate the strategic use of
CPT-1 inhibitors in certain clinical situations. For example, such
agents might still prove useful in the acute reversal of diabetic
ketoacidosis (63), in the prevention of myocardial injury or
arrhythmia during reperfusion of the heart after ischemia (64),
or in the treatment of congestive heart failure (65).
ACKNOWLEDGMENTS
R.L.D. was supported by a Career Development Award from
the American Diabetes Association and a Development Partners
Junior Faculty Award from SmithKline-Beecham. Additional
support was received from Novo-Nordisk Pharmaceuticals,
the National Institutes of Health (DK-18573), and the Forrest
C. Lattner Foundation.
128 DIABETES, VOL. 50, JANUARY 2001
MUSCLE LIPID AND INSULIN RESISTANCE
FIG. 5. Poor correlation between glucose disposal and whole-body
fat mass in rats receiving low-fat and lard diets either alone or sup-
plemented with the CPT-1 inhibitor, etomoxir (Eto). Each value rep-
resents the mean ± SE for five to eight determinations.
We are indebted to Murphy Daniels, Evelyn Babcock,
and Gary Binkley for their excellent technical assistance on
this project.
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