Marcadores de Utilização da Gordura em Humanos

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Markers of capacity to utilize fatty acids in human
skeletal muscle: relation to insulin resistance and
obesity and effects of weight loss
JEAN-AIMÉ SIMONEAU,*,1 JACQUES. H. VEERKAMP,† LORRAINE P. TURCOTTE,‡ AND
DAVID E. KELLEY§,2
*Division of Kinesiology, Department of Social and Preventive Medicine, Faculty of Medicine, Laval
University, Ste-Foy, Quebec; †Department of Biochemistry, Faculty of Medical Sciences, University of
Nijmegen, Nijmegen, The Netherlands; ‡Department of Exercise Sciences, University of Southern
California, Los Angeles, California; and Division of Endocrinology and Metabolism, and §Department
of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA
ABSTRACT A number of biochemical defects have
been identified in glucose metabolism within skeletal
muscle in obesity, and positive effects of weight loss
on insulin resistance are also well established. Less is
known about the capacity of skeletal muscle for the
metabolism of fatty acids in obesity-related insulin
resistance and of the effects of weight loss, though it
is evident that muscle contains increased triglycer-
ide. The current study was therefore undertaken to
profile markers of human skeletal muscle for fatty
acid metabolism in relation to obesity, in relation to
the phenotype of insulin-resistant glucose metabo-
lism, and to examine the effects of weight loss.
Fifty-five men and women, lean and obese, with
normal glucose tolerance underwent percutaneous
biopsy of vastus lateralis skeletal muscle for deter-
mination of HADH, CPT, heparin-releasable (Hr)
and tissue-extractable (Ext) LPL, CS, COX, PFK, and
GAPDH enzyme activities, and content of cytosolic
and plasma membrane FABP. Insulin sensitivity was
measured using the euglycemic clamp method.
DEXA was used to measure FM and FFM. In skeletal
muscle of obese individuals, CPT, CS, and COX
activities were lower while, conversely, they had a
higher or similar content of FABPC and FABPPM than
in lean individuals. Hr and Ext LPL activities were
similar in both groups. In multivariate and simple
regression analyses, there were significant correla-
tions between insulin resistance and several markers
of FA metabolism, notably, CPT and FABPPM. These
data suggest that in obesity-related insulin resistance,
the metabolic capacity of skeletal muscle appears to
be organized toward fat esterification rather than
oxidation and that dietary-induced weight loss does
not correct this disposition.—Simoneau, J.-A.,
Veerkamp, J. H., Turcotte, L. P., Kelley, D. E.
Markers of capacity to utilize fatty acids in human
skeletal muscle: relation to insulin resistance and
obesity and effects of weight loss. FASEB J. 13,
2051–2060 (1999)
Key Words: carnitine palmitoyl transferase z fatty acid binding
proteins z lipoprotein lipase z maximal aerobic power z fatty
acid oxidation
Skeletal muscle has an important role not only for
the utilization of glucose during insulin stimulated
conditions, but also for the metabolism of fatty acids
(FA); indeed, during fasting conditions, oxidation of
FA is generally considered to be the principal sub-
strate of skeletal muscle (1). Numerous biochemical
alterations of skeletal muscle and insulin signaling
pathways have been identified in relation to insulin-
resistant glucose metabolism, as well as improve-
ments after weight loss (2, 3). As recently stated (4),
however, in obesity the capacity of skeletal muscle for
the metabolism of FA has been less clearly delin-
eated. Activity of heparin releasable lipoprotein
lipase (Hr LPL) has been reported to be lower or
equivalent in skeletal muscle of nondiabetic over-
weight compared to normal weight subjects (5, 6);
among a relatively small group of obese women, our
laboratories found reduced activity of carnitine
palmitoyl transferase (CPT) in muscle (7). Skeletal
muscle capacity for FA utilization is potentially im-
portant both in relation to systemic fat balance and
insulin resistance. In regard to insulin resistance, fat
content within muscle is a strong correlate and has
been found to be increased in obesity (8–12). The
mechanisms that cause skeletal muscle lipid content
to increase in obesity are unclear, but might be
broadly postulated as an increased uptake of lipids,
1 Dr. Jean-Aime Simoneau passed away on August 27, 1999,
after a lengthy illness. His deep commitment, and indeed
passion, for scientific inquiry will be sorely missed. In a career
that was too brief, he made outstanding and original contri-
butions to our understanding of muscle biochemistry and of
its importance to obesity and diabetes.
2 Correspondence: East-1140 Biomedical Science Tower,
University of Pittsburgh, Pittsburgh, PA 15213, USA. E-mail:
Kelley@msx.dept.-med.pitt.edu
20510892-6638/99/0013-2051/$02.25 © FASEB
decreased oxidation, or a combination of both pro-
cesses.
During insulin-stimulated conditions, FA oxida-
tion in skeletal muscle is normally suppressed (13),
yet incomplete suppression of FA occurs in obesity-
related insulin resistance (14, 15). Nevertheless, it
might be incorrect to infer from this that FA oxida-
tion is always increased within skeletal muscle in
obesity. Instead, during fasting conditions, when FA
oxidation normally is predominant within skeletal
muscle, the rate of oxidation in skeletal muscle has
been reported to be reduced in obesity and Type 2
diabetes (16, 17). A prospective clinical study re-
vealed that reduced FA oxidation is a metabolic risk
factor for weight gain (18) and that enzyme activities
within skeletal muscle pertaining to lipid metabolism
might contribute to lower FA oxidation (19, 20).
Moreover, after weight loss, skeletal muscle in post-
obese individuals may continue to be inefficient in
the oxidation of fat (21, 22). It has also been found
that aerobic exercise training can augment skeletal
muscle capacity for FA oxidation and uptake (23–25)
and that exercise training induces higher levels of
activity for muscle aerobic-oxidative enzymes (26).
Reduced activity of oxidative enzymes in skeletal
muscle has been found in obesity and in relation to
insulin resistance (10, 27–29). Recent findings of
increased skeletal muscle content of malonyl CoA
and diacylglycerol in animal models of obesity and
insulin resistance suggest one mechanism (30, 31).
Therefore, a biochemical basis for impaired FA
oxidation within skeletal muscle in obesity is cer-
tainly plausible, but it has not been yet clearly
established.
The current study was undertaken to compare
lean and obese individuals for markers of skeletal
muscle capacity to utilize FA and to examine the
effects of weight loss on these markers in obese
subjects. We postulated that in obesity-related insulin
resistance, skeletal muscle would manifest a reduced
capacity for FA oxidation. Also, to examine whether
patterns of muscle enzyme activity and other key
proteins of fat metabolism were interrelated to the
phenotype of insulin resistance, glucose metabolism
was determined using the euglycemic insulin infu-
sion method.
MATERIALS AND METHODS
Subjects
Fifty-five healthy adults (28 women and 27 men) participated
in this study. Forty-five subjects were white, nine were African-
American, and one volunteer was Asian-American. All poten-
tial volunteers underwent a medical examination, including
routine laboratory testing and a 75 g oral glucose tolerance
test prior to inclusion. Those individuals with glucose intol-
erance, hyperlipidemia, or liver, renal, or thyroid disorders
were excluded as were individuals with a history or physical
findings of coronary heart disease, peripheral vascular dis-
ease, hypertension, or neuromuscular illnesses. Subjects tak-
ing medication for these disorders or taking medications
known to influence carbohydrate and lipid metabolism were
also excluded from participation. An additional inclusion
criterion was that both lean and obese volunteers not be
engaged in a program of regular physical exercise, so as to
minimize differences of physical fitness as a confounding
variablebetween the two groups. Physical activity patterns
were determined by interview at the time of screening. The
protocol was approved by the University of Pittsburgh Insti-
tutional Review Board and all volunteers gave written in-
formed consent.
Body composition and VO2max
As a measure of physical fitness, maximal aerobic power
(VO2max) was measured using an incremental, modified
Bruce protocol. A mass spectrometer (Marquette Electronics,
Milwaukee, Wis.) was used to analyze expired air for CO2 and
O2 fractions during this test; hemodynamic parameters were
measured prior to, during, and immediately after this test.
Whole body fat mass (FM) and fat-free mass (FFM) were
assessed by dual energy X-ray absorptiometry (DEXA; Lunar
model DPX-L, Madison, Wis.), using transverse scans to
measure fat and lean tissue mass.
Insulin sensitivity and muscle biopsy
On the evening prior to measurement of insulin sensitivity
and performing the percutaneous muscle biopsy, subjects
were admitted to the University of Pittsburgh General Clinical
Research Center. That evening, they received a standard
dinner (10 kcal/kg; 50% carbohydrate, 30% fat, 20% pro-
tein) and then fasted overnight. Subjects were instructed to
consume a weight-maintaining diet containing at least 200 g
carbohydrate and to avoid exercise or strenuous exertion for
at least 3 preceding days; all subjects had maintained a stable
weight for at least 3 months prior to the initial studies. At ;
7:00 am, subjects were moved to the metabolic assessment
suite and a catheter was placed in a forearm vein for infusion
of glucose, insulin, and a primed (20 mCi), continuous (0.20
mCi/min) infusion of high performance liquid chromatogra-
phy-purified [3-3H]-glucose (New England Nuclear, Boston,
Mass.); the latter was for measurement of glucose utilization
during the final 40 min of the insulin infusion. Isotope was
started 100 min before the insulin infusion. A percutaneous
muscle biopsy of the vastus lateralis muscle was done after 60
min of bed rest and 30 min prior to starting insulin infusion
(32). Muscle samples were immediately frozen in liquid N2
and kept at 280°C for later assays. Insulin was then infused at
40 mU/min-m2 body surface area for 3 h and euglycemia was
maintained using an adjustable infusion of 20% dextrose, to
which 80 mCi of [3-3H]-glucose had been added to maintain
stable plasma glucose specific activity (33). Plasma glucose
was determined at 5 min intervals during the clamp. Blood
samples for measurement of [3-3H]-glucose specific activity
were collected every 10 min during the final 40 min of insulin
infusion.
Weight loss intervention
Subjects with a body mass index greater than 30 kg/m2 were
invited to participate in a 4 month, out-patient weight loss
program. The goal of the weight loss intervention was to
produce a weight loss of ;10–15 kg. The weight loss program
combined a very low-calorie diet (VLCD) with an intensive
2052 Vol. 13 November 1999 SIMONEAU ET AL.The FASEB Journal
program of behavioral intervention, as described previously
(34), and was initiated immediately after baseline physiolog-
ical assessments. Briefly, during the first 10 wk of this pro-
gram, subjects were instructed to consume a very low-calorie
diet (800 kcal/day), followed by 6 wk of gradual refeeding up
to isocaloric requirements. During the VLCD, subjects con-
sumed a combination of liquid formula (Optifast, Novartis
Corporation) and lean meat, fish, and fowl. During weeks
11–13, subjects were instructed to consume 1200 kcal/day,
with a gradual reintroduction of fruits, vegetables, and grains
to the diet. During weeks 14–16, subjects were instructed to
consume a weight-maintaining diet, with 30% of calories as
fat, 15% as protein, and 55% as complex carbohydrates, in
order to avoid effects of acute fasting or calorie restriction on
post-weight loss physiological assessments. All subjects under-
went metabolic reassessments at week 17, after 3 wk of this
weight-maintenance diet. Subjects were seen weekly for 16 wk
by a nutritionist/behaviorist and serum potassium was moni-
tored at 2 wk, and then repeated with an EKG, blood
chemistries, and uric acid at 4 wk intervals. Vitamin and
mineral supplementation were provided during the VLCD.
To avoid potential confounding effects of exercise on muscle
biochemistry, subjects were carefully instructed to maintain
pre-weight loss levels of physical activity. After this weight loss
intervention, reassessments of body composition, VO2max,
and insulin sensitivity were performed and a second percuta-
neous muscle biopsy of vastus lateralis, immediately adjacent
to the site of the initial biopsy, was obtained.
Analysis
Plasma glucose was measured using an automated glucose
oxidase reaction (YSI 2300 Glucose Analyzer, Yellow Springs,
Ohio). Glucose-specific activity was determined with liquid
scintillation spectrometry after the deproteinization of
plasma with barium sulfate and zinc hydroxide. Serum insulin
was determined using a commercially available radioimmu-
noassay kit (Pharmacia, Uppsala, Sweden). Rates of plasma
glucose appearance (Ra) and utilization (Rd) were calculated
using the Steele equations, as modified for variable rate
glucose infusions containing isotope (33).
For the determination of enzyme activity in skeletal muscle,
small pieces of the muscle sample (;10 mg) were homoge-
nized in a glass-glass Duall homogenizer with 39 vol. of
ice-cold extracting medium (0.1 M Na-K-phosphate, 2 mM
EDTA, pH57.2). Homogenate was transferred into 1.5 ml
polypropylene tubes; this suspension was magnetically stirred
on ice for 15 min and sonicated six times for 5 s at 20 watts,
on ice, with pauses of 85 s between pulses. The resulting
homogenate was used for determination of activity (Vmax)
of six enzymes: citrate synthase (CS; EC 4.1.3.7), cytochrome
c oxidase (COX; EC 1.9.3.1), phosphofructokinase (PFK;
EC 2.7.1.11), glyceraldehyde phosphate dehydrogenase
(GAPDH; EC 1.2.1.12), beta-hydroxyacyl CoA dehydrogenase
(HADH; EC 1.1.1.35), and CPT (EC 2.3.1.21). Spectrophoto-
metric techniques were conducted at 30°C, according to
methods previously used (7, 16, 35, 36). Enzyme activities
were expressed in Units (micromoles of substrate per
minute) per gram of wet weight tissue (U/g). For determina-
tion of skeletal muscle activity of lipoprotein lipase (LPL; EC
3.1.1.34), ;10 mg of muscle tissue was weighed and incu-
bated for 45 min at 37°C in 0.5 ml of Krebs-Ringer (131 mM
NaCl, 5 mM KCl, 1 mM KH2PO4, 0.7 mM CaCl2, and 1 mM
MgSO4), mixed with 0.1M Tris-HCl (pH 8.6) buffer contain-
ing 1% FFA-free bovine serum albumin (Sigma, St. Louis,
Mo.), and 7 U of heparin (Sigma) in order to extract the
heparin-releasable fraction of muscle LPL (37). Heparin
eluate was kept on ice and the tissue was transferred to a Duall
tissue grinder and homogenized with 50 volumes of 223 mM
Tris-HCl buffer (pH 8.6) containing 0.25M sucrose, 5 mM
sodium deoxycholate, 0.08% Triton X-100, heparin (9
U/ml), and 1% bovine serum albumin to measure the
extractable fraction of muscle LPL. One hundred microliter
aliquots of the Hr eluates and 75 ml aliquots of the Ext
fraction were incubated in triplicate with 100 ml of 14C
triolein (100 mCi/ml) and triolein (7 mM), which were
sonicated in 5% gum arabic, 75 mM Tris-HCl (pH 8.6), 75
mM NaCl, 0.2% bovine serum albumin, and 10% human
serum. After 2 h of incubation at 37°C under agitation,
reaction was stopped by the addition of 3.25 ml of an
extraction mixture containing chloroform:methanol:heptane
(141:125:100) and 1.05 ml of 50 mM potassium carbonate
borate hydroxide buffer (pH 10.0) (38). After mixing, sam-
ples were centrifuged at 1500 3 g for 15 min and 2.0 ml of the
upper aqueous phase containing FA was added to 10 ml of a
scintillation mixture (Aquasol) and counted in a beta counter
(LKB-Wallac). LPL activity was expressed as nanomoles of
FFA transformed/min/g of wet weight muscle.
FABPPM and FABPC content within skeletal muscle
Homogenates of muscle samples were prepared to measure
cytosolic(FABPc) and plasma membrane (FABPpm) fatty acid
binding protein contents. Ten micrograms of total proteins
(determined according to the Bradford technique) were
deposited on a 6% stacking gel and migrated through a 12%
resolving gel for 120 min at 100 V. After migration, gels were
electrically transferred onto PVDF membranes at 100 V for
120 min. These membranes were blocked with a 5% pow-
dered milk solution, rinsed thrice in TTBS solution (Tris: 10
mM; NaCl: 150 mM; Tween: 0.05%), and incubated 1 h with
either a polyclonal antibody developed from a rat hepatic
FABPpm (10 ml/20 ml of TTBS) or a polyclonal antibody
developed from a human heart FABPc (5 ml/50 ml of TTBS).
After further washing with TTBS, the antibody–antigen com-
plex was incubated with a goat anti-rabbit IgG (1 ml/50 ml of
TTBS for FABPpm and 5 ml/50 ml of TTBS for FABPc) for 1 h.
Membranes were washed and stained with an alkaline phos-
phatase staining kit (BCIP/NBT) following the recommenda-
tions of the manufacturer (Promega, Madison, Wis.). The
single band detected on the blots corresponded to the
expected molecular mass for FABPc (14 kDa) and FABPpm
(40 kDa). Although mitochondrial aspartate aminotransfer-
ase protein is recognized with the FABPpm antibody, the effect
of this detection is minimal in the present study, since almost
90 to 95% of the FABPpm contained in the total homogenate
has been shown to be from the skeletal muscle plasma
membrane (39). Standard amounts of a human latissimus
dorsi muscle homogenate or pure FABPc served as internal
control on each membrane and the integrated density of
stained bands was measured twice using the NIH Image
analysis software.
Statistical analysis
Data are presented as mean 6 se, unless otherwise indicated.
Two-way analysis of variance (ANOVA) was used to examine
for effects of group (obese vs. lean) and gender with respect
to muscle markers, body composition, and insulin sensitivity.
Repeated measures ANOVA was used to examine for the
effect of weight loss on these measures. Linear regression and
stepwise multiple regression were used to detect a correlation
among adiposity, VO2max, insulin sensitivity, and muscle
markers. Statistics were performed using Sigma Stat 2.0
(SPSS, Chicago, Ill.).
2053MARKERS OF FATTY ACID METABOLISM IN HUMAN MUSCLE
RESULTS
Body composition and insulin sensitivity
Clinical characteristics of body composition and
values for insulin-stimulated glucose metabolism and
VO2max are shown in Table 1 (changes in these
parameters among the cohort who completed the
weight loss intervention are subsequently shown in
Table 3). Compared with lean subjects, those who
were obese had reduced insulin sensitivity, lower
VO2max, and greater FM. There were some gender-
related differences. Though matched for BMI,
women had a higher percentage of FM than did men
and had lower mean VO2max. As previously reported
in this cohort of subjects (11), there were significant
negative correlations between insulin sensitivity and
obesity (BMI and FM).
Influence of obesity on skeletal muscle markers of
glycolysis and oxidative and fatty acid metabolic
pathways
Within vastus lateralis skeletal muscle, obese subjects
had higher activity for the two marker enzymes of
glycolysis (PFK and GAPDH) and somewhat lower
activities for marker enzymes of oxidative capacity
(CS, COX, and HADH), as shown in Table 2. The
glycolytic to oxidative ratio (PFK/CS), an integrative
parameter of skeletal muscle metabolic differentia-
tion (40), was higher in obesity (4.160.9 vs. 6.560.5
for lean and obese women, and 6.060.8 vs. 7.060.5
for lean and obese men).
Levels of muscle FABPs were either similar or
increased in obese compared to lean subjects. The
FABPpm content of muscle was ;30% higher in
obese compared to lean men, whereas it was almost
60% higher in obese women compared to their lean
TABLE 1. Clinical characteristics of lean and obese men and womena
Women Men
Lean (n 5 7) Obese (n 5 21) Lean (n 5 8) Obese (n 5 19)
Age (yrs) 35 6 2 37 6 1 34 6 1 37 6 1
BMI (kg/m2) 22.9 6 1.3 33.5 6 0.7 24.0 6 1.2 34.3 6 0.8
FFM (kg) 40.7 6 2.9 48.5 6 1.7 57.2 6 2.7 70.1 6 1.8
FM (kg) 18.9 6 2.9 38.3 6 1.7 13.2 6 2.7 34.8 6 1.8
(% wt) (28.9 6 2) (42.2 6 1.2) (18.0 6 1.9) (31.8 6 1.2)
Rd (mg/min-kg FFM) 9.9 6 0.8 6.5 6 0.5 8.9 6 0.8 5.2 6 0.5
VO2 max (ml/min-kg FFM) 42.5 6 1.5 36.9 6 0.9 43.8 6 1.4 42.6 6 0.9
a All variables were significantly (P , 0.01) different between obese and lean and between men and women, except age and the absence
of gender effects on BMI and Rd.
TABLE 2. Skeletal muscle markers in lean and obese men and womena
Women Men
Obesity GenderLean Obese Lean Obese
Glycolytic metabolism
PFK 38.7 6 5.3 55.3 6 3.1 62.6 6 4.7 63.3 6 3.2 0.05 0.001
GAPDH 363 6 26 428 6 14 486 6 21 520 6 15 0.01 0.001
Oxidative metabolism
CS 9.8 6 1.1 8.8 6 0.7 12.0 6 1.0 9.8 6 0.7 0.08 0.08
COX 8.6 6 0.9 7.2 6 0.5 8.8 6 0.7 7.6 6 0.5 0.05 NS
HADH 18.3 6 1.5 16.1 6 0.8 15.8 6 1.2 14.5 6 0.8 0.13 0.07
Fatty acid metabolism
FABPpm 25.5 6 5.8 41.8 6 2.2 32.5 6 4.5 42.1 6 2.4 0.01 NS
FABPc 84.8 6 10.9 80.2 6 4.9 46.0 6 8.2 62.7 6 5.1 NS 0.001
Hr LPL 5.83 6 0.08 4.16 6 0.33 2.67 6 0.83 4.33 6 0.05 NS 0.08
Ext LPL 6.00 6 2.17 3.83 6 0.66 3.83 6 1.33 4.50 6 0.66 NS NS
CPT 0.13 6 0.01 0.11 6 0.01 0.15 6 0.01 0.11 6 0.01 0.01 NS
a Activities of phosphofructokinase (PFK), glyceraldehyphosphate dehydrogenase (GAPDH), citrate synthase (CS), cytochrome c oxidase
(COX), beta-hydroxyacyl CoA dehydrogenase (HADH), and carnitine palmitoyl transferase (CPT) are expressed in U/g wet weight muscle.
Heparin releasable (Hr) and extractable (Ext) lipoprotein lipase (LPL) activities are expressed in nmoles of FFA transformed per min per gram
of wet weight muscle. Cytosolic fatty acid bind protein (FABPc) is expressed in micrograms/g wet weight muscle while plasma membrane fatty
acid protein is expressed in arbitrary units per g wet weight muscle.
2054 Vol. 13 November 1999 SIMONEAU ET AL.The FASEB Journal
counterpart. Obese men also had a greater content
within muscle for FABPc than did lean men, but
there was a significant gender difference: women
had significantly greater content of FABPc in skeletal
muscle compared to men; among women, no differ-
ence for FABPc was observed in regard to obesity.
Another marker related to capacity of skeletal mus-
cle for the uptake of FA is activity of Hr LPL, which
did not differ in lean and obese subjects, nor did
activity of Ext LPL.
Although the levels of FABPs and LPL activity
indicate that capacity of skeletal muscle for taking up
FA or for cytosolic binding is not impaired in obesity,
the activity of the CPT complex, regarded as rate-
limiting for entry of long-chain fatty acylCoA esters
into mitochondria, was lower in obesity (P,0.01).
Across the cohort of subjects, CPT activity in skeletal
muscle was positively correlated with activity of CS
(r 50.55, P,0.001), COX (r 50.60, P,0.001) and
with HADH (r 50.51, P,0.001), indicating that its
activity was correlated to markers of oxidative en-
zyme capacity within skeletal muscle. To discern
whether the reduced activity of CPT in obesity was
more pronounced than the general pattern of re-
duced oxidative capacity, the CPT:COX ratio was
calculated. There was no difference for the CPT:
COX ratio between lean and obese subjects (nor
when other oxidative marker enzymes were used in
the denominator), suggesting that decreased skeletal
muscle activity of CPT in obesity appears to be
proportionate to reduced activity of oxidative en-
zymes and, indirectly, to the mitochondrial content
of muscle.
To explore further the concept that the muscle
capacity for FA uptake might be differentially
regulated from its capacity for FA oxidation in
obesity, the ratio of CPT:FABPpm was calculated.
The CPT:FABPpm ratio was significantly lower in
obese compared to lean individuals (P,0.001), as
shown in Fig. 1.
Correlation of muscle markers with obesity and
insulin resistance
In addition to group differences based on categori-
cal definition ofobese vs. non-obese status, data on
markers of lipid metabolism were examined for
correlation with phenotypes of insulin sensitivity and
adiposity. There was a modest negative correlation
between PFK:CS ratio and insulin sensitivity (r
520.37, P,0.01), indicating the skeletal muscle
with heightened glycolytic capacity relative to its
oxidative enzyme capacity manifests insulin resis-
tance. Several markers of FA metabolism also corre-
lated with insulin sensitivity. Muscle CPT activity was
positively correlated with insulin sensitivity (r 50.34,
P,0.01) whereas muscle FABPpm was negatively re-
lated to insulin sensitivity (r 520.33, P,0.05), as was
activity of Ext LPL (r 520.39, P,0.01). FABPc con-
tent within muscle did not correlate significantly
with insulin sensitivity. Because the ratio of CPT:
FABPpm was lower in obesity, its relation to insulin
sensitivity was examined and a significant positive
correlation was observed (r 50.43, P,0.01). More-
over, the CPT:FABPpm ratio was significantly and
negatively correlated with the PFK:CS ratio (r
520.46, P,0.001), suggesting that the perturbation
between the glycolytic and oxidative capacity is inter-
related to an imbalance between uptake and mito-
chondrial oxidative capacities for FA metabolism.
Effects of weight loss on FA, glycolytic, and
oxidative metabolic capacities of skeletal muscle
Thirty-two obese subjects completed the 4 month
weight loss program and a post-weight loss (post-WL)
metabolic assessment. Data on body composition
and insulin sensitivity are shown in Table 3. After
weight loss, there were significant decreases in adi-
posity and a significant improvement in insulin sen-
sitivity. One of the goals of our intervention was to
isolate effects of dietary-induced weight loss and not
introduce effects of exercise training, at least in an
uncontrolled manner. This goal was met as values for
VO2max were unchanged after weight loss and were
within a range associated with sedentary lifestyles.
The metabolic data from pre- and post-WL muscle
biopsies are shown in Table 4.Only very few gender
differences appeared in the response to body weight
Figure 1. The ratio of CPT activity within vastus lateralis
skeletal muscle to that of FABPpm was determined for 15 lean
subjects, 40 obese subjects, and for 32 of the obese subjects
who were studied after weight loss. There was a highly
significant difference between lean and obese subjects
(P,0.001) and no significant effect of weight loss to change
the value of this ratio.
2055MARKERS OF FATTY ACID METABOLISM IN HUMAN MUSCLE
loss intervention. Glycolytic enzyme activities in skel-
etal muscle (GAPDH and PFK) did decrease after
weight loss. The activity of CS in skeletal muscle did
not change after weight loss, whereas COX and
HADH activities decreased significantly in women,
but not in men. Activities of muscle CPT as well as of
Hr and Ext LPL did not change in men or women.
Muscle content of FABPc and of FABPpm were not
altered by weight loss. There were no changes in the
ratio of glycolytic to oxidative enzyme capacity (PFK/
CS) or in the ratio of CPT:FABPpm (Fig. 1) in men
and women after weight loss.
DISCUSSION
Obesity can cause insulin resistance of skeletal mus-
cle, whereas even moderate amounts of weight loss
can improve insulin sensitivity. The current study,
which entailed both a cross-sectional comparison of
lean and obese individuals and weight loss interven-
tion, reaffirms these principles while addressing a
more novel concept, that the levels of metabolic
markers of skeletal muscle for the utilization of FA
are altered in obesity in a manner that favors lipid
accumulation. Skeletal muscle, owing to its size and
importance in the regulation of systemic energy
expenditure (41), has a key role in the utilization of
both carbohydrate and lipid substrates (13). Several
metabolic steps might contribute to an impaired
reliance on FA oxidation within skeletal muscle
during fasting conditions. In a separate report of the
postabsorptive rates of FFA uptake and oxidation
from the participants in this study based on values
measured with the use of the arteriovenous leg
balance technique, key findings in obesity were that
fasting patterns of fatty acid metabolism within skel-
etal muscle favor lipid esterification rather than
oxidation (42). The goal of the current investigation
was to assess whether there were obesity-related
perturbations in the activity or content of metabolic
markers that play a role either in the entrance of FA
through the membrane of the muscle cell, in intra-
cellular transport, in entrance of FA into mitochon-
dria, and in the capacity for FA oxidation by mito-
chondria. Novel findings reported in the current
study demonstrate that a number of biochemical
markers of the capacity of skeletal muscle to metab-
olize FA are altered in obesity.
Numerous FABPs have been described in recent
years (24, 43, 44). Some are thought to be involved
in the transmembrane movement of FA, but so far
there have been relatively few studies in human
skeletal muscle. Although the exact role of the
TABLE 3. Effects of weight loss on body composition, insulin sensitivity, and maximal aerobic powera
Women Men
Pre-WL Post-WL Pre-WL Post-WL
Weight (kg) 92.5 6 2.9 80.3 6 2.8* 108.9 6 3.5 91.4 6 2.7*
Fat mass (kg) 39.1 6 1.4 29.6 6 1.5* 34.6 6 2.5 22.3 6 1.7*
Rd (mg/min-kg FFM) 6.2 6 0.7 7.3 6 0.7* 5.7 6 0.8 8.1 6 0.8*
VO2 max(ml/min-kg FFM) 36.6 6 1.0 36.4 6 1.0 42.1 6 1.1 44.0 6 1.5
a Significantly different than pre-weight loss values; *P , 0.05.
TABLE 4. Skeletal muscle markers in lean and obese men and women before and after weight loss
Women Men
Pre-WL Post-WL Pre-WL Post-WL
Glycolytic metabolism
PFK 56 6 2 54 6 2 64 6 3 55 6 4*
GAPDH 428 6 16 371 6 14* 511 6 15 415 6 15*
Oxidative metabolism
CS 8.7 6 0.4 7.9 6 0.3 10.1 6 0.8 10.7 6 0.8
COX 7.1 6 0.4 6.1 6 0.4* 7.8 6 0.5 7.9 6 0.4
HADH 15.9 6 0.9 14.0 6 0.9* 14.7 6 1.0 14.8 6 1.0
Fatty acid metabolism
FABPpm 43.2 6 1.9 39.5 6 1.9 45.2 6 2.0 44.2 6 2.1
FABPc 79.0 6 6.0 78.0 6 6.0 67.0 6 6.0 69.0 6 9.0
Hr LPL 5.83 6 1.33 4.17 6 0.33 2.66 6 0.83 4.33 6 0.50
Ext LPL 6.00 6 2.17 3.83 6 0.66 3.83 6 1.33 4.50 6 0.66
CPT 0.11 6 0.01 0.10 6 0.07 0.11 6 0.11 0.11 6 0.01
* Significant differences between pre- and post-weight loss values; P , 0.05. Abbreviations are in Table 2.
2056 Vol. 13 November 1999 SIMONEAU ET AL.The FASEB Journal
plasma membrane fatty acid binding protein has not
been completely elucidated, recent evidence has
shown that it may be an important component of a
fatty acid transport system in muscle. It was recently
shown that fatty acid binding to plasma membrane
proteins and fatty acid uptake by giant sarcolemmal
vesicles in muscle saturate with an increase in un-
bound fatty acid concentration and are substantially
inhibited by the presence of antibodies to FABPPM
(45, 46). The uptake data are an important piece of
evidence, because when using giant sarcolemmal
vesicles, uptake reflects only cellular transport rather
than cellular transport and metabolism. Thus, the
presence of saturation for fatty acid uptake and of
inhibition by the antibodies to FABPPM suggests that
a fatty acid transport system exists in muscle and that
FABPPM plays a role in this transport system (24, 45,
46). In the present study, FABPpm content was in-
creased in obesity in both men and women and also
correlated with the phenotype of insulin-resistant
glucose metabolism. These data on FABPpm suggest
that skeletal muscle in obese individuals maintains at
least an equivalent and perhaps an enhanced capac-
ity for the entrance of FA within the muscle cell.
Lipoprotein lipase, implicated in the processes
of FA entrance through its action as a triglyceride
hydrolase within the muscle cell (47– 49), has
received particular attention in obesity studies.
This enzyme within muscle exists as two metabol-
ically active fractions; each represents approxi-
mately half of total LPL activity in human muscle
(50), proportions that were confirmedby the Hr
and Ext LPL activities assayed in the current study.
The Hr LPL is considered to be present at the
capillary endothelium and active in hydrolyzing
triglycerides within lipoproteins (48). Although
the cellular control of intramuscular (i.m.) triglyc-
eride metabolism presumably involves two major
identified lipases— hormone sensitive lipase and
LPL—the Ext LPL was proposed to serve to replen-
ish enzyme at the capillary surface or to be in-
volved in the hydrolysis of intracellular triglyceride
in skeletal muscle (51). In the current study, Hr
LPL activity was not different between lean and
obese subjects and was not correlated with mea-
sures of body composition or insulin sensitivity.
These results agree with prior observations in
nondiabetic obese and non-obese individuals (6),
though some conflicting data have also been re-
ported (5, 52, 53). However, Ext LPL activity was
increased in relation to insulin resistance. If the
concept is correct that Ext LPL is involved in i.m.
triglyceride lipolysis, then its content within mus-
cle might contribute to insulin resistance in a
causal manner by elevating the intracellular con-
tent of triglycerides within the muscle cell. Further
research is needed to specifically examine this
concept, particularly in view of the increased fat
content that has been described in the myocytes of
obese individuals (12).
Other novel information reported here is the
determination of the muscle cytosolic 14 kDa FABP.
FABPc is a relatively abundant cytosolic protein, and
although our current understanding of its precise
physiological role(s) has not advanced as far as
knowledge of structure, it can viewed as a molecule
that provides niches for hydrophobic binding of fatty
acids and long-chain acyl CoA esters within cells
(43). Accordingly, it may serve to protect the internal
milieu from adverse effects of “free’ FA and perhaps
subserve some directional trafficking of lipid sub-
strates. Absolute values of FABPc obtained in the
present study are similar to those previously reported
by Van Nieuwenhoven et al. (54). Although a com-
parison is not always possible, cytosolic FABP data
have also been published for rat muscles; the values
reported have ranged from 13 6 1 (superficial and
white portion of quadriceps) to 303 6 24 (soleus)
mg/g of wet weight muscle (55). The current study
demonstrates that the content of FABPc within skel-
etal muscle is normal in obesity. Among our cohort
of obese and non-obese subjects, no correlation
between insulin sensitivity and FABPc was observed,
though women had a significantly greater content of
FABPc within muscle. Although FABPc content did
not differ between lean and obese women, there was
a significantly higher content in obese compared to
lean men. This increased amount of that protein in
muscle may be without major consequence on the
processes of FA oxidation, however, since no effect
on rates of FA oxidation capacity was observed in a
diabetic animal model with overexpression of FABPc
in skeletal muscle (56).
To test whether there was an imbalance between
the capacity for the uptake and binding of FA within
the cytosol and the mitochondrial enzyme capacity
for FA oxidation, the activity of CPT, the enzyme
complex regarded as a rate-limiting step for the
entrance and oxidation of long-chain fatty acid CoA
esters (57, 58), and two markers of the mitochon-
drial oxidative capacity (CS and COX) were mea-
sured. Confirming previous findings obtained in a
smaller number of women (7), there was a reduction
in muscle CPT activity. This lower CPT activity in
obesity was proportionate to a reduction in CS, an
enzyme of the Krebs cycle, and COX, one of the
regulatory step of the respiratory chain, findings
again confirming previous data from our laborato-
ries (59). Considering that studies carried out pre-
dominately in animal models of insulin resistance
and obesity reported increased muscle content of
malonyl CoA, a strong allosteric inhibitor of CPT I
(31), a decreased level of CPT combined with an
increased malonyl CoA content would create a favor-
2057MARKERS OF FATTY ACID METABOLISM IN HUMAN MUSCLE
able intracellular metabolic condition for a reduced
or impaired capacity for FA oxidation within skeletal
muscle.
Borrowing from the concept of the glycolytic to
oxidative enzyme ratio, a ratio that has been used to
characterize the metabolic differentiation of skeletal
muscle (40) and has been shown by us to be strongly
related to insulin resistance (10), we examined the
ratio between CPT activity and the content of FAB-
Ppm as a means of integrating into a single value the
biochemical capacities for FA binding and entrance
at the level of the plasma membrane of the muscle
cell and mitochondrial oxidation. In obesity, the
CPT:FABPpm ratio is reduced to one-half the value
found in skeletal muscle of lean individuals. More-
over, this ratio correlated with the phenotype of
insulin resistance. The inference we derive from the
CPT:FABPpm ratio is that in obesity, skeletal muscle
appears to be equipped to take up FA from plasma
but has a disproportionately reduced capacity for
their oxidation. As an increased triglyceride content
within skeletal muscle occurs in obesity and is found
to be a relatively strong marker of insulin resistance,
our findings seem to provide a potential biochemical
mechanism for an enhanced disposition for esterifi-
cation of FA.
Cross-sectional studies cannot fully clarify whether
a decrease in the metabolic capacities within muscle
of obese subjects occurs as a consequence of obesity
or, instead, might add to the risk of becoming obese.
In the current study, a weight loss intervention was
used to address this issue. Regrettably, we found that
weight loss, at least weight loss as induced by dietary
intervention, does not rectify the obesity-related met-
abolic derangements of skeletal muscle, suggesting
that the observed perturbations most likely do not
develop as a consequence of obesity. Among the
eight different markers of the skeletal muscle aero-
bic-oxidative and FA metabolism investigated in the
current study, neither CPT activity nor the activity or
content of the other markers, except the reduction
in COX and HADH activities in women, were mod-
ified after weight loss. There is undoubtedly a need
to determine whether interindividual variations in
these skeletal muscle phenotypes are under a strict
control of genetic factors or whether they can be
altered by strategies other than dietary restriction. At
least in lean individuals, exercise training not only
enhances insulin sensitivity, but also increases the
capacity of skeletal muscle for FA oxidation (23, 50).
This suggests that as exercise induces metabolic
fitness in skeletal muscle, this entails enhanced ca-
pacities for both glucose and FA utilization, and that
in sedentary obese individuals the ‘metabolic unfit-
ness’ of skeletal muscle entails a combined impaired
of both glucose and FA metabolic pathways. The
failure of weight loss to improve oxidative capacity of
skeletal muscle, including activity of CPT, despite its
positive effects on insulin sensitivity, could demar-
cate a biochemical risk factor for the recidivism of
weight gain, which unfortunately is quite common
after dietary-induced weight loss.
In summary, the metabolic capacities of skeletal
muscle ascertained in obese subjects within the cur-
rent study form an overall profile characterized by an
imbalance between the capacity for the uptake and
transport of FA within the muscle cell and a reduced
capacity of their oxidation within the mitochondria.
These altered patterns correlate with the phenotype
of insulin resistance in weight stable obese individu-
als; yet unlike glucose metabolism, which improves
after weight loss, the biochemical markers of FA
metabolism were essentially unaffected by dietary
restriction and remained different from muscle of
lean individuals. This composite of muscle metabolic
markers further strengthens the concept that the
mitochondrialbioenergetic capacities of skeletal
muscle are perturbed in human obesity and may
contribute both to the expression of insulin-resistant
patterns of glucose metabolism and in partitioning
fat toward esterification within muscle.
The cooperation of our research volunteers is gratefully
acknowledged. We appreciate the skill and cooperation of the
nursing and nutritional staff at the General Clinical Research
Center and particularly the contribution of Dr. Rena Wing at
the Obesity and Nutrition Research Centers of the University
of Pittsburgh. We wish to specifically acknowledge the efforts
of Nancy Mazzei, Bret Goodpaster, Yves Gélinas, and Michel
Lacaille of Laval University and the secretarial support of
Nancy Penny. This project was supported by funding from
National Institutes of Health R01 DK49200–02,
5M01RR00056 (General Clinical Research Center), and
1P30DK46204 (Obesity and Nutrition Research Center). We
also acknowledge the support of Novartis Pharmaceuticals,
which provided the Optifast for the weight loss intervention,
and the Zumberge Research and Innovation Fund of the
University of California.
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Received for publication November 17, 1998.
Revised for publication April 22, 1999.
2060 Vol. 13 November 1999 SIMONEAU ET AL.The FASEB Journal