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/m2), 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-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 Student’s t-test for independent samples was used to compare groups. Correlations were tested using Pearson’s 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 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.

Table 1. General and metabolic characteristics of the impaired glucose tolerant subjects (IGT) and normal glucose tolerant controls (NGT).

Mean ± sem. Student’s 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.

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.

Fig. 1. Skeletal muscle protein content (µg/g wet weight) of CD36 (1A) and FABPc (1B) during fasting and after insulin stimulation in obese men. Open diamonds are normal glucose tolerant controls; filled circles are impaired glucose tolerant subjects; flat object with dotted line is the mean. * P < 0.05, paired Student’s t-test, two-tailed.

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. Interestingly, a positive association was found between the increase in CD36 protein and GIR (Pearson r = 0.564, p = 0.045). Correction for a possible confounding by baseline values, by dividing the change in CD36 protein content by fasting CD36 protein content, did not reduce the association (r = 0.640, p = 0.020). Although this finding seems contra-intuitive, it has been shown that in insulin resistance, CD36 protein translocation is impaired (20), showing an increased amount of CD36 at the sarcolemma and a reduced translocation after insulin stimulation in vitro. A larger increase in CD36 protein may be a mechanism to compensate for a reduced translocation effect. On the other hand, an increased fatty acid transporter capacity during the time that meal-derived LCFA are highly available from chylomicrons and VLDL is likely to increase intramyocellular lipid storage, and thus these subjects may have become more insulin resistant.

In conclusion, CD36 protein is regulated in a remarkably dynamic manner by insulin in vivo in human skeletal muscle of obese subjects. This is a promising finding because an increase in CD36 protein content in the late postprandial phase may play an important role in an increased fractional extraction of fatty acids from plasma, enhancing intramyocellular triglyceride storage. It also emphasises that the uptake of free fatty acids may be regulated at the level of skeletal muscle by fatty acid transporters and not only by plasma lipid supply. In addition, our data suggest that this regulation may depend on the degree of insulin resistance.

Acknowledgements: Antibody MO25 was kindly provided by Dr. N. N. Tandon, Otsuka America Pharmaceutical, Inc., Rockville, MD, USA. We thank our volunteers without whom this study would not have been possible. We thank Jos Stegen, Gert Schaart, Joan Senden, Eveline Peeters-Tielen, Judith Huskens and Dorien Mintjes for their excellent assistance. Supported by grants from the Dutch Diabetes Research Foundation (DFN 98.901 and DFN 2000.00.020). JFCG is Netherlands Heart Foundation Professor of Cardiac Metabolism.


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