Insulin acutely upregulates protein expression of the fatty acid
transporter CD36 in human skeletal muscle in vivo
Citation for published version (APA):
Corpeleijn, E., Pelsers, M. M. A. L., Soenen, S., Mensink, M., Bouwman, F. G., Kooi, M. E., Saris, W. H. M.,
Glatz, J. F. C., & Blaak, E. E. (2008). Insulin acutely upregulates protein expression of the fatty acid transporter
CD36 in human skeletal muscle in vivo.
Journal of Physiology and Pharmacology
,
59
(1), 77-83.
Document status and date:
Published: 01/01/2008
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E. CORPELEIJN
1
, M. M.A.L. PELSERS
2
, S. SOENEN
1
, M. MENSINK
1
, F. G. BOUWMAN
1
,
M. E. KOOI
3
, W. H.M. SARIS
1
, J. F.C. GLATZ
4
, E. E. BLAAK
1
INSULIN ACUTELY UPREGULATES PROTEIN EXPRESSION
OF THE FATTY ACID TRANSPORTER CD36 IN HUMAN SKELETAL
MUSCLE IN VIVO.
1
From the Department of Human Biology, the Nutrition and Toxicology Research Institute
Maastricht (NUTRIM), Maastricht University, Maastricht, The Netherlands;
2
Department
of movement sciences, the Nutrition and Toxicology Research Institute Maastricht NUTRIM,
Maastricht University, Maastricht, The Netherlands;
3
the Department of Radiology, University
Hospital Maastricht, Maastricht, The Netherlands;
4
Department of Molecular Genetics,
Cardiovascular Research Institute Maastricht (CARIM), Maastricht University,
Maastricht, The Netherlands
Enhanced fatty acid uptake may lead to the accumulation of lipid intermediates. This
is related to insulin resistance and type 2 diabetes mellitus. Rodent studies suggest
that fatty acid transporters are acutely regulated by insulin. We investigated
differences in fatty acid transporter content before and at the end of a
hyperinsulinemic euglycemic clamp in skeletal muscle (m. vastus lateralis) of obese,
glucose-intolerant men (IGT) and obese normal glucose tolerant controls (NGT).
The fatty acid transporter FAT/CD36 protein content increased 1.5-fold (P < 0.05)
after 3-hrs of insulin stimulation with no difference between IGT and control
subjects. No change was seen in cytosolic fatty acid binding protein (FABPc) protein
content. The increase in FAT/CD36 protein content was positively related to insulin
resistance as measured during the clamp (r = 0.56, P < 0.05). An increase in
FAT/CD36 protein content in skeletal muscle may result in a higher fractional
extraction of fatty acids (larger relative uptake) after a meal, enhancing triglyceride
accumulation in the muscle. We conclude that also in obese humans the FAT/CD36
protein content in skeletal muscle is dynamically regulated by insulin in vivo on the
short term.
Keywords: skeletal muscle, obesity, impaired glucose tolerance, lipid metabolism,
FAT/CD36, insulin action
JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2008, 59, 1, 7783
www.jpp.krakow.pl
INTRODUCTION
An imbalance between elevated plasma long-chain fatty acid (LCFA)
availability, uptake and oxidation results in intramyocellular accumulation of
LCFA metabolites, such as fatty acyl-CoA, ceramides, and diacylglycerol (1-4).
Elevated levels of these LCFA metabolites are likely to induce defects in the
insulin signalling cascade and are associated with the development of skeletal
muscle insulin resistance and type 2 diabetes (1-5). Impaired utilization is not
only reported in type 2 diabetic patients (6), but also in subjects with impaired
glucose tolerance (7, 8), a prediabetic state, suggesting that impaired fatty acid
utilization may be an important early factor in the development of type 2 diabetes.
It is not clear under which metabolic conditions the accumulation of triglycerides
in skeletal muscle takes place, but increased storage can be due to increased
circulating concentrations of LCFA and triglycerides (TG), as well as to an
impaired suppression of plasma LCFA after a meal (9). Previously, it has been
shown in type 2 diabetic patients that triglycerides can accumulate after high fat
meals during the day (10). Not only an increased supply of lipids, but also an
increased fractional extraction (relative uptake) of LCFA (plasma LCFA or TG-
derived LCFA) can enhance the accumulation of fatty acids. Fatty acid
transporters play a critical role in fractional fatty acid uptake, in particular when
the fatty acid: albumin ratio is low, as is the case after a meal (11, 12). Indeed, the
fatty acid transporter CD36 is sensitive to insulin, and a recent study in
cardiomyocytes has shown that insulin can rapidly, within hours, increase CD36
mRNA expression as well as protein content, which contributed to an increased
fatty acid uptake capacity (13).
MATERIALS AND METHODS
Nine obese men with impaired glucose tolerance (IGT) and eight obese men with normal
glucose tolerance (NGT), matched for age and BMI, participated in the study. Inclusion criteria
were obesity (BMI > 30 kg/m
2
), diastolic blood pressure < 100 mm Hg, no major health problems,
and no use of medication that could influence the measurements. The NGT men had no family
history of diabetes. Subjects were screened for glucose metabolism with a standard oral glucose
tolerance test (75 g glucose) with capillary blood sampling at baseline and after 2 hrs. Subjects were
included according to the WHO criteria of 1999 for capillary plasma (IGT: fasting < 7.0 mmol/l,
2hr postload > 8.9 and < 12.2 mmol/l). Three subjects with glucose values (fasting < 8.0 mmol/l
and 2hr postload < 14.8 mmol/l) above the cutoff points were included as well. The experimental
protocol was approved by the local Medical Ethical Committee of the Maastricht University. All
subjects gave written informed consent.
The NGT and IGT men underwent measurements for body composition using hydrostatic
weighing, aerobic capacity using an incremental exhaustive bicycle test and insulin sensitivity using
a hyperinsulinemic euglycemic clamp (1 mU*kg BW
-1
*min
-1
). The glucose infusion rate (GIR,
mmol glucose/min) per kg fat free mass (FFM) was determined during a steady state of 30 min.
after at least 120 min of insulin infusion. Muscle biopsies were taken before and after insulin-
78
stimulation at the end of the steady state of the clamp, freed from any visible fat and blood and
immediately frozen in liquid nitrogen or for immunofluorescence in isopentane at its melting point.
Muscle type FABPc was measured by means of ELISA (Hycult Biotechnology, Uden, the
Netherlands) (14), while CD36 protein was analysed with a in-house developed sandwich-type
ELISA (15). Biopsy lipid content was analysed using Oil Red O staining (16). Slides were
incubated with a primary antibody against adult human slow myosin heavy chain (A4.951,
Developmental Studies Hybridoma Bank, Iowa City, USA) to determine fibre type and a rabbit
polyclonal antiserum against human laminine (pLam, Sigma) to visualize myocyte membranes.
Images were captured using a Nikon E800 fluorescence microscope (Uvikon, Bunnik, the
Netherlands) and a colour CCD camera (Basler A101 C) with 240 times magnification. Per biopsy,
at least 50 different cells were analyzed using Lucia 5.49 software.
Plasma FFA and glucose were analyzed in EDTA plasma using standard enzymatic techniques
automated on the COBAS Fara centrifugal analyzer (for FFA: FFA-C test kit, Wako chemicals,
Neuss, Germany; for glucose: Roche Unikit III, Hoffman-la-Roche, Basel, Switzerland). Insulin
was analyzed using a fluoroimmunometric assay (autoDELFIA Insulin, PerkinElmer, Turku,
Finland) with no cross-reactivity with proinsulin or split forms of proinsulin.
Results are given as mean ± sem. A two-tailed Students t-test for independent samples was
used to compare groups. Correlations were tested using Pearsons correlation coefficient (r). P <
0.05 was considered statistically significant. Statistical analysis was performed using SPSS 10.0 for
Macintosh.
RESULTS AND DISCUSSION
No differences in CD36 or FABPc content were found between the obese IGT
men and obese controls (Table 1). Skeletal muscle CD36 protein increased 1.5
fold after 3 hours of insulin-stimulation (p < 0.05, figure 1A), the change in CD36
protein content was comparable between NGT and IGT subjects (p = 0.62, Fig.
1A). Two men (one IGT and one NGT) showed a decrease. In contrast, skeletal
muscle FABPc protein content did not change (p = 0.22, Fig. 1B). The rapid
increase in CD36 protein content indicates that the uptake of plasma LCFA into
skeletal muscle may be actively regulated by fatty acid transporters at the level of
skeletal muscle itself, and not only in a passive way by plasma lipid supply.
Insulin directly activates glucose transporters, but also appears to activate fatty
acid transport. This can be very relevant, considering that LCFA from
chylomicrons and VLDL may become available for uptake in a later stage after
meal intake (9). Indeed, Chabowski and coworkers found the same remarkable
dynamic upregulation of CD36 protein content, already after 1 hour of insulin
stimulation in rat cardiomyocytes (13). This was preceded by an increase in
mRNA expression. Insulin also induced the translocation of CD36. In that study,
a large part of the newly synthesized CD36 protein was translocated to the plasma
membrane, suggesting that the new proteins may directly contribute to the fatty
acid uptake capacity of the muscle cell. Also in humans, insulin induces the
translocation of CD36 to the plasma membrane in response to insulin infusion
(17). Apparently, insulin has two fast effects: within minutes, it induces the
translocation of endosomal CD36 protein to the sarcolemma, and within hours
79
this is followed by an increase in total CD36 protein, which is also immediately
available for translocation. Both adaptations lead to more sarcolemmal CD36 and
an increased fatty acid uptake capacity after a meal. This may be an important
adaptation for a rapid storage of meal-derived fatty acids.
It is remarkable that despite the increase in CD36 protein, we did not find an
increase in muscle FABPc. If the muscle increases its fatty acid uptake capacity,
would it then not be necessary to also increase the intracellular capacity to
transport fatty acids? Studies with FABPc knock-out mice have indicated the
involvement of FABPc in shuttling LCFA from the sarcolemma to intracellular
sites of oxidation or esterification, but rather in a permissive than in a regulatory
fashion (18, 19). Even a reduction of FABPc protein of 50% is sufficient to
maintain LCFA trafficking. Thus, in comparison to CD36, the need to increase
FABPc protein content is limited.
The increase in CD36 protein content upon insulin stimulation (Fig. 1A) was
comparable between groups (p = 0.62). The change in CD36 protein in relation
to insulin resistance was further investigated in the group as a whole.
80
Table 1. General and metabolic characteristics of the impaired glucose tolerant subjects (IGT) and
normal glucose tolerant controls (NGT).
NGT IGT P-value
n = 9 n = 8
Age (yrs) 57.1 ± 2.6 58.1 ± 2.7 0.79
Capillary glucose fasting (mmol/l) 5.7 ± 0.6 6.8 ± 1.0 0.035
Capillary glucose 2-hour OGTT (mmol/l) 6.7 ± 1.2 12.97 ± 1.6 0.001
Body mass index (kg/m
2
) 34.2 ± 1.5 32.6 ± 0.6 0.28
Body fat (%) 34.7 ± 1.5 32.7 ± 1.1 0.51
Fat free mass (kg) 69.5 ± 10.8 62.7 ± 3.8 0.084
Waist-hip ratio 1.02 ± 0.02 1.03 ± 0.01 0.59
VO
2
max (ml O
2
* kg FFM
-1
*min
-1
) 40.5 ± 2.0 38.9 ± 1.8 0.57
Triglycerides (mmol/l) 1.73 ± 0.29 1.54 ± 0.27 0.65
Glucose - fasting (mmol/l) 5.7 ± 01 6.2 ± 0.2 0.059
Glucose - SS (mmol/l) 4.5 ± 0.1 4.4 ± 0.1 0.40
Insulin - fasting (mmol/l) 16.8 ± 4.1 14.1 ± 1.8 0.092
Insulin - SS (mmol/l) 111 ± 6 108 ± 9 0.76
FFA - fasting (mmol/l) 539 ± 54 696 ± 81 0.64
FFA - SS (mmol/l) 140 ± 19 167 ± 13 0.27
GIR (µmol*kgFFM
-1
*min
-1
) 32.2 ± 4.5 21.2 ± 3.7 0.085
muscle CD36 protein (ng/g wet weight) 20.0 ± 4.1 16.5 ± 4.3 0.57
muscle FABPc protein (µg/g wet weight) 93.6 ± 13.6 83.5 ± 13.8 0.61
IMTG (Oil Red O, lipid stained area fraction) 0.058 ± 0.034(n=5) 0.068 ± 0.041(n=4) 0.85
Fibre type area (% type 1 fibre) 46.5 ± 8.7 (n=5) 51.3 ± 11.6 (n=4) 0.75
Mean ± sem. Students t-test for unpaired samples, two-tailed. FFM = fat free mass, GIR = glucose
infusion rate, IGT = impaired glucose tolerance, IMTG = intramyocellular triglycerides, NGT =
normal glucose tolerance, SS = steady state (last half hour) during insulin-stimulation (clamp). n =
9 for NGT and n = 8 for IGT unless indicated otherwise.