Departments of 1 Human Biology, 2 Physiology, 3 Internal Medicine, 4 Nuclear Medicine, and 5 Anesthesiology, Maastricht University and Academic Hospital, 6200 MD Maastricht, The Netherlands
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ABSTRACT |
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In this study, we
investigated the hypothesis that impairments in forearm skeletal muscle
free fatty acid (FFA) metabolism are present in patients with type 2 diabetes both in the overnight fasted state and during -adrenergic
stimulation. Eight obese subjects with type 2 diabetes and eight
nonobese controls (Con) were studied using the forearm balance
technique and indirect calorimetry during infusion of the stable
isotope tracer [U-13C]palmitate after an overnight fast
and during infusion of the nonselective
-agonist isoprenaline (Iso,
20 ng · kg lean body mass
1 · min
1). Additionally, activities of mitochondrial enzymes
and of cytoplasmatic fatty acid-binding protein (FABP) were determined
in biopsies from the vastus lateralis muscle. Both during fasting and
Iso infusion, the tracer balance data showed that forearm muscle FFA uptake (Con vs. type 2: fast 449 ± 69 vs. 258 ± 42 and Iso
715 ± 129 vs. 398 ± 70 nmol · 100 ml
tissue
1 · min
1, P < 0.05) and FFA release were lower in type 2 diabetes compared with Con.
Also, the oxidation of plasma FFA by skeletal muscle was blunted during
Iso infusion in type 2 diabetes (Con vs. type 2: Iso 446 ± 274 vs. 16 ± 70 nmol · 100 ml tissue
1 · min
1, P < 0.05). The net forearm
glycerol release was increased in type 2 diabetic subjects
(P < 0.05), which points to an increased forearm
lipolysis. Additionally, skeletal muscle cytoplasmatic FABP content and
the activity of muscle oxidative enzymes were lowered in type 2 diabetes. We conclude that the uptake and oxidation of plasma FFA are
impaired in the forearm muscles of type 2 diabetic subjects in the
overnight fasted state with and without Iso stimulation.
type 2 diabetes; skeletal muscle; free fatty acids; fatty acid-binding protein; stable isotopes
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INTRODUCTION |
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OBESITY AND TYPE 2 DIABETES frequently occur together, indicating that these two conditions may share common pathological mechanisms. Increased circulating plasma free fatty acid (FFA) concentrations have been indicated as an important risk factor in the etiology of both conditions and in the predisposition toward other chronic diseases like cardiovascular disease (9). An increased delivery of FFA to the liver may underlie many metabolic disturbances in obesity and type 2 diabetes like a decreased insulin binding to hepatocytes (24), a diminished (hepatic) insulin clearance (12), an impaired insulin-mediated suppression of hepatic glucose output (15), and an increased very low density lipoprotein (VLDL)-triacylglycerol output (9). Additionally, it has been proposed that chronically elevated FFA concentrations may reduce insulin secretion in type 2 diabetes (17).
On one hand, the elevated FFA concentrations may result from an
increased release from the expanded adipose tissue stores (13) and impaired insulin-mediated suppression of
lipolysis (18). On the other hand, defects in muscle FFA
uptake exist in obesity and type 2 diabetes (2,
3, 5, 14). Previously, we have
shown a diminished skeletal muscle uptake of FFA in (abdominally) obese
subjects during -adrenergic stimulation (2,
3), suggesting that the sympathetic nervous system may be
of importance in the disturbances in fat utilization. Arteriovenous
difference measurements across leg muscle of Kelley and co-workers
indicate that there may be a defect in uptake and oxidation of plasma
FFA in the postabsorptive state in (visceral) obesity (5)
and in obesity-associated type 2 diabetes (14). Because of
its large total mass and increased energy expenditure during physical
activity, skeletal muscle is one of the most important organs involved
in FFA metabolism in healthy humans (6), indicating that a
diminished muscle utilization of fatty acids may lead to an increase in
the plasma FFA concentrations, especially under conditions with a high
lipolytic rate (starvation, exercise, adrenergic stimulation).
The aim of the present study was, first, to investigate whether the
observed impairment in muscle fat metabolism in type 2 diabetes
(14) also extends to catecholamine-stimulated conditions. Second, we intended to better define previous findings on an increased muscle respiratory quotient in type 2 diabetes (14) by
measuring the oxidation of plasma-derived fatty acids at the forearm
muscle and whole body level. Third, muscle biopsies of the vastus
lateralis were taken to determine indicators of fatty acid transport,
-oxidation, and general oxidative capacity.
We clearly want to indicate that it is not the aim of the present study to investigate the relative importance of obesity and type 2 diabetes in the development of the observed impairment in fat metabolism. Follow-up research will be needed to answer this aspect.
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SUBJECTS AND METHODS |
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Eight healthy lean males and eight obese male subjects with type
2 diabetes (diabetes duration 2 yr, range 0.5-8 yr) were studied.
The diabetic subjects were treated with diet alone (n = 2) or with diet in combination with oral blood glucose-lowering agents
(n = 6 were treated with low doses of sulfonylureas,
which were withheld for 2 days before the experiments). Beside this, no
other medication was used. None of the subjects had serious health
problems apart from their diabetes. A normal resting electrocardiogram and blood pressure was a prerequisite for participation. Subject characteristics are indicated in Table 1.
All subjects engaged no more than 3 h/wk in sports, and none had a
physically demanding job. The study protocol was approved by the
Medical Ethical Review Committee of Maastricht University, and all
subjects gave written informed consent.
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Body Composition
Body weight was determined on an electronic scale, accurate to 0.1 kg. Waist and hip circumference measurements to the nearest 1 cm were made with the subject standing upright. Body composition was determined by hydrostatic weighing with simultaneous lung volume measurement (Volugraph 2000, Mijnhardt, The Netherlands). Body composition was calculated according to Siri (23).Experimental Design
All subjects participated in two experiments in random order, with 2 wk in between. Protocol 1 was performed to determine FFA kinetics during baseline and during intravenous infusion of the nonselectiveOn a separate occasion, needle muscle biopsies were taken percutanously
from the vastus lateralis muscle after an overnight fast, using the
Bergström method with suction (8). The biopsies were
immediately frozen in liquid nitrogen and stored at 80°C until the
determination of the activities of mitochondrial enzymes, lactate
dehydrogenase (LDH), and cytoplasmatic fatty acid-binding protein
(FABP; see Biochemical Methods).
Protocol 1. Whole body and forearm skeletal muscle metabolism were studied by means of the forearm muscle balance technique during continuous intravenous infusion of the stable isotope tracer [U-13C]palmitate.
Before the start of the experiment, three canulas were inserted. One canula was inserted under local anesthesia in the radial artery of the forearm for sampling of arterial blood. In the same arm, a second canula was inserted in a forearm vein for the infusion of Iso and the stable isotope tracer. In the contralateral arm, a third catheter was inserted in retrograde direction in an antecubital vein for the sampling of deep venous blood, draining forearm muscle. Measurements were done during the last 30 min of a 90-min baseline period (0-90 min) and a subsequent 60 min period of intravenous infusion of the nonselectiveProtocol 2.
The acetate recovery in expired air was determined in a separate
experiment during a 90-min baseline period and a 60-min period of Iso
infusion. After collection of a background breath sample, subjects
received an intravenous priming dose of 0.085 mg/kg body wt
NaH13CO3. Next, a continuous infusion of
[1,2-13C]acetate was started and continued during the
entire period. The acetate tracer (sodium salt of
[1,2-13C]acetate, 99% enriched; Cambridge Isotope
Laboratories) was dissolved in 0.9% saline to obtain a 3.11 ± 0.16 mM solution. Acetate was infused at a rate of 0.088 µmol
· kg body wt1 · min
1 to obtain the
same 13C infusion rate per time unit as for the palmitate
tracer. Breath samples were taken at similar time points as during the
palmitate infusion.
Biochemical Methods
Blood samples were taken simultaneously from the radial artery and deep forearm vein after the blood flow measurement while the hand circulation was still occluded. Duplicate 1-ml blood samples were immediately injected with a needle through the rubber stopper of preweighed vacutainer tubes, without disturbing the vacuum, for the determination of 13CO2/12CO2. After being weighed again, 1 vol of 1 M sulfuric acid (H2SO4) was injected through the rubber stopper in the tubes to direct all blood CO2 in the gaseous head space. The tubes were weighed again to determine the dilution factor. The gaseous head space was finally brought to atmospheric pressure with helium. The same procedure was applied to bicarbonate standards of known concentration. The coefficient of variation of this method is 0.4% for CO2 concentrations in the range of 15-30 mM. The coefficient of variation between duplicate measurements of CO2 concentrations is 0.09%.All other blood samples were collected in EDTA-containing tubes on ice
and were immediately centrifuged at 4°C, and the plasma was put in
liquid nitrogen until storage at 80°C.
Breath samples and blood samples were analyzed for the 13C-to-12C ratio and CO2 content by injecting 20 µl of the gaseous head space in a gas chromatography (GC) continuous-flow isotope ratio-mass spectrometer (Finnigan MAT 252).
For the determination of plasma palmitate and total FFA kinetics, FFA were extracted from plasma, isolated by TLC, and derivatized to their methyl esters. Isotope enrichment of palmitate was determined by GC-isotope ratio mass spectrometry after on-line combustion of the fatty acids to CO2.
Palmitate concentrations were determined on an analytical GC with ion flame detection using heptadecanoic acid as internal standard, and, on average, it comprised 24.5 ± 0.6% of total FFA concentration. Total plasma FFA, glucose, glycerol, lactate, and infusate acetate were measured using standard enzymatic techniques automated on the Cobas Fara centrifugal analyzer at 340 nm (for FFA: FFA-C test kit; Wako Chemicals, Neuss, Germany; for glucose: Roche Unikit III, Hoffman-La Roche, Basel, Switzerland; for glycerol and lactate: Boehringer, Mannheim, Germany). Plasma insulin was measured using a specific double-antibody RIA for human insulin (Kabi Pharmacia, Uppsala, Sweden). Also, the hematocrit was determined in heparinized blood using a microcapillary system.
The muscle biopsies were homogenized in ice-cold Tris-EDTA buffer at pH 7.4. The homogenates were subsequently sonicated for 4 × 15 s and were centrifuged at 10,000 g for 2 min at 4°C to remove cell debris. Tissue content of heart-type or muscle-type cytoplasmatic FABP in skeletal muscle was measured by means of a newly developed ELISA, using recombinant human FABP as standard (27). Citrate synthase (CS) was determined by the method of Sherpherd and Garland (20), whereas 3-hydroxyacyl-CoA dehydrogenase (HAD) and LDH activities were assayed according to Bergmeyer (1).
Calculations
Blood flow.
SMBF was calculated according to the following equation
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Gas exchange monitoring.
The metabolic rate was calculated from the rates of O2
uptake (O2) and CO2
production (
CO2) according to the
equation of Weir (26). Carbohydrate and fat oxidation
rates were calculated from
O2 and
CO2 and urinary nitrogen excretion
(9).
Tracer calculations.
The palmitate flux (rate of appearance or rate of disappearance) was
calculated as
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Forearm muscle.
The exchange of metabolites across forearm muscle was calculated by
multiplying the arteriovenous concentration difference of
metabolites (µmol/l) by forearm plasma flow (ml · 100 ml
forearm muscle1 · min
1) or by TBF
(for CO2 exchange). Forearm plasma flow was calculated by
multiplying forearm blood flow by (1
hematocrit)/100. A
positive exchange indicates uptake.
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Statistical Analysis
Data are expressed as means ± SE. To compare baseline and Iso-induced responses between groups, a two-factor repeated-measures ANOVA was performed. A P value <0.05 was regarded as statistically significant. ![]() |
RESULTS |
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Tracer Enrichment in Blood and Expired Air
The plasma palmitate concentration and the 13C-to-12C ratio of plasma palmitate did not change significantly during the sampling times from 60 to 90 min during the baseline period and from 30 to 60 min of Iso infusion in both groups, allowing the applications of steady-state equations for the tracer data (data not shown). The acetate recovery (Fig. 1) was significantly lower in type 2 diabetes compared with controls, and the increase in this variable throughout the experiment was significantly blunted in type 2 diabetes. Palmitate recovery followed the same pattern (at 90 min: controls 8.0 ± 0.3 vs. type 2 diabetes 6.0 ± 0.3% and at 150 min: 12.0 ± 0.4 vs. 9.3 ± 0.7%, P < 0.001) and was on average threefold lower than acetate recovery.
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Arterial Hormone and Substrate Concentrations
Arterial glucose, lactate, insulin, and glycerol concentrations were significantly higher in type 2 diabetes both during basal conditions and during Iso infusion, whereas FFA concentrations were not significantly different between groups (Table 2). Basal insulin concentrations and the Iso-induced increase in insulin concentrations were higher in type 2 diabetes compared with control (P < 0.01).
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Whole Body Energy Expenditure and Substrate Utilization
After 20 min of Iso infusion, a steady state was reached for O2 exchange, CO2 exchange, and energy expenditure. Resting energy expenditure and the Iso-induced thermogenesis (%increase above baseline: control 13.7 ± 2.2 vs. type 2 diabetes 14.4 ± 1.3%) were comparable in both groups (Table 3). Resting fat oxidation tended to be higher (P = 0.11), whereas resting carbohydrate oxidation was lower (P < 0.05) in type 2 diabetic subjects compared with controls. The Iso-induced increase in fat oxidation was less pronounced in type 2 diabetes compared with controls (P = 0.05). Furthermore, Iso infusion led to a decrease in carbohydrate oxidation in controls, whereas there was a slight increase in type 2 diabetes (P < 0.05).
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The mean FFA flux per kilogram LBM was not significantly different between groups both during baseline conditions and Iso infusion (Table 3).
Plasma FFA oxidation was not significantly different between both groups during baseline, whereas the Iso-induced increase in plasma FFA oxidation was significantly blunted in type 2 diabetes (P < 0.001, Table 3). The proportion of the FFA flux that was oxidized was similar during baseline conditions in both groups (controls 39 ± 2 and type 2 diabetes 40 ± 3%), whereas the Iso-induced decrease in this variable was significantly more pronounced in type 2 diabetes compared with controls (controls 38 ± 1 and type 2 diabetes 34 ± 2%, P < 0.05).
Forearm Muscle Metabolism
Forearm composition.
The total forearm area was significantly higher in type 2 diabetes
(control vs. type 2 diabetes 5,840 ± 132 vs. 7,039 ± 430 mm2, P < 0.05). When expressed as the
percentage of total forearm area, the percentages of skin (5.9 ± 0.1 vs. 5.4 ± 0.2%, P < 0.05) and muscle
(66.8 ± 1.0 vs. 61.3 ± 1.0%, P < 0.001)
were significantly lower in type 2 diabetes, whereas the amount of
subcutaneous adipose tissue was significantly higher in type 2 diabetes
(13.5 ± 1.1 vs. 20.9 ± 1.2%, P < 0.001).
Baseline TBF and the Iso-induced increase in TBF (P < 0.001) tended to be lower in type 2 diabetes (Table
4), but differences between groups did
not reach statistical significance. Forearm subcutaneous ATBF also
significantly increased during Iso infusion in both groups
(P < 0.001), and there were no differences between
both groups (baseline vs. Iso: control 2.40 ± 0.49 vs. 4.22 ± 1.04 and type 2 diabetes: 1.82 ± 1.04 vs. 5.29 ± 1.23 ml · 100 g tissue1 · min
1).
Relative values for SBF were comparable in both groups and were similar
during baseline and Iso-stimulated conditions (baseline vs. Iso:
control 5.2 ± 0.6 vs. 5.1 ± 0.3 and type 2 diabetes: 5.0 ± 0.5 vs. 4.8 ± 0.5 ml · 100 g
tissue
1 · min
1). Estimated SMBF and
TBF were not significantly different in controls and type 2 diabetes
(Table 4). It can be concluded that changes in TBF are similar to
changes in SMBF. For this reason, TBF (or PBF) is used in calculating
skeletal muscle substrate exchange.
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Forearm muscle substrate exchange. Deep venous O2 saturation of Hb was 40.0 ± 2.7% in controls and 43.9 ± 2.6% in type 2 diabetes, indicating that the forearm substrate exchange mainly reflects muscle metabolism. Net muscle glucose uptake was significantly lower in type 2 diabetes compared with controls both under basal conditions and during Iso infusion (Table 4). In both groups, there was an increase in muscle lactate release during Iso infusion. Both during baseline and during Iso infusion, muscle glycerol release was higher in type 2 diabetes, whereas net muscle FFA uptake tended to be lower in type 2 diabetes compared with controls. The net plasma FFA uptake by the forearm is two- and sixfold higher than FFA delivery derived from forearm lipolysis (3 times glycerol release) in controls during overnight-fasted conditions and Iso stimulation, respectively, whereas in type 2 diabetic subjects net FFA uptake is more than twofold lower than FFA delivery derived from forearm lipolysis in both conditions.
Components of forearm muscle FFA metabolism.
Both during basal conditions and Iso infusion, skeletal muscle FFA
uptake and FFA release were significantly lowered in type 2 diabetes
(Fig. 2). Expressing the contribution of
muscle FFA uptake to whole body rate of disappearance of FFA yields
significantly lower values in type 2 diabetes compared with controls
(on average: type 2 diabetes 10% vs. control 15%; Fig.
3).
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Skeletal muscle biopsies.
Cytoplasmatic FABP content of the vastus lateralis muscle was
significantly higher in controls than in type 2 diabetic subjects (Table 5). Also, muscle oxidative
capacity, reflected by the activity of HAD and CS, was lowered in type
2 diabetes, whereas LDH activity was comparable in both groups.
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DISCUSSION |
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We have used an isotopic tracer approach in combination with the
forearm balance technique to quantify the uptake, release, and
oxidation of plasma FFA by skeletal muscle in obesity-associated type 2 diabetes during postabsorptive and catecholamine-stimulated conditions.
The present data show a reduced uptake of plasma FFA by skeletal muscle
in obese subjects with mild type 2 diabetes under baseline conditions
and during intravenous infusion of the nonselective -agonist Iso.
Additionally, during Iso infusion, the oxidation of plasma FFA by
skeletal muscle was significantly increased in controls (accounting for
50% of muscle FFA uptake), whereas in type 2 diabetic subjects
muscle 13CO2 production was not significantly
different from zero. The impairments in skeletal muscle plasma
fatty acid uptake and oxidation are accompanied by a diminished
skeletal muscle content of cytoplasmatic FABP and a diminished muscle
oxidative capacity.
Methodological Considerations
Forearm muscle blood flow. The results of the present study show that changes in TBF are similar to changes in SMBF in both control and type 2 diabetic subjects, indicating that it is valid to use TBF (or forearm plasma flow) for calculating forearm skeletal muscle substrate fluxes, as reported previously in obese subjects (2).
Acetate recovery factor. To correct the rate of plasma FFA oxidation for the incomplete label recovery in breath, an acetate recovery factor was used; the recovery factor has been proposed to correct for label fixation of a fatty acid tracer in pathways going from the entrance of labeled acetyl-CoA to the tricarboxylic acid cycle until the recovery of label in expired CO2. This factor has been extensively validated in experiments of Sidossis et al. (21, 22) and more recently by Schrauwen et al. (19) for experiments lasting <2 h in lean and obese subjects. Additionally, the acetate recovery factor across (leg) muscle has been measured and was reported to be about equal to the whole body recovery factor (16).
Baseline Fatty Acid Utilization
During baseline conditions, there was an impaired uptake of FFA by muscle in type 2 diabetic subjects, resulting in a significant decrease in the contribution of skeletal muscle to whole body FFA disposal (10% in type 2 diabetes vs. 15% in control; Fig. 3). These data seem to correspond to data of Kelley and co-workers (5, 14), showing an impaired muscle FFA uptake during basal conditions in visceral obesity and type 2 diabetes.In the present study, there was no significant forearm muscle 13CO2 production under overnight-fasted conditions both in controls and in type 2 diabetic patients, suggesting no oxidation of plasma FFA by skeletal muscle. However, we cannot exclude the possibility that the 13CO2 production cannot be measured during the 90-min baseline infusion period with the present tracer infusion rate, as the recovery of 13CO2 from a palmitate tracer may be low (after 90 min infusion: 7% recovery in expired air) and may increase linearly in time for periods of 5-6 h. Thus, to draw definite conclusions on the quantitative differences in the oxidation of plasma fatty acids by skeletal muscle between patients and controls during overnight-fasted conditions, a higher infusion rate and/or a more prolonged infusion period may be required.
Iso-Mediated Fatty Acid Utilization
As during baseline conditions, we found a diminished forearm muscle FFA uptake in the type 2 diabetic subjects during Iso-stimulated conditions, resulting in an impaired systemic FFA clearance by skeletal muscle. Additionally, in type 2 diabetic subjects, muscle 13CO2 production was not significantly different from zero during Iso infusion, whereas the oxidation of plasma FFA by skeletal muscle was significantly increased in controls (accounting for 50% of muscle FFA uptake). In accordance with the forearm muscle data, the increases in the whole body oxidation of plasma FFA and total fat oxidation were significantly blunted in type 2 diabetes compared with controls. Thus these data strongly point to an impairment of Iso-induced fat oxidation in type 2 diabetes.Underlying Mechanisms
Forearm muscle glycerol release was three and five times as high in type 2 diabetic subjects compared with controls during basal and Iso-stimulated conditions, respectively (Table 4). On one hand, this may be explained by a higher intramuscular or plasma VLDL triglyceride lipolysis and/or a higher lipolysis in adipose cells in the perimysium covering muscle, which indicates, in view of the lowered FFA release, an increased utilization (oxidation and/or reesterification) of FFA released by forearm lipolysis in type 2 diabetic subjects. Increased forearm lipolysis in fact may flood the muscle with FFA and reduce the FFA concentration gradient between blood and muscle, which is one of the primary determinants of plasma FFA uptake and oxidation (25). Therefore, an increased forearm lipolysis may be an important mechanism for the reduced uptake and oxidation of plasma FFA. However, on the other hand, care has to be taken in interpreting muscle glycerol metabolism from net release data as glycerol tracer balance studies have shown that glycerol disappears into muscle and may be metabolized (7). Therefore, the increased forearm glycerol release in type 2 diabetic subjects also may be a consequence of a decreased rate of intramuscular glycerol metabolism. Further studies are necessary to elucidate this issue.To examine other potential mechanisms behind the lowered plasma FFA utilization by skeletal muscle in type 2 diabetes, FABP and activities of mitochondrial enzymes were examined. Muscle biopsy analyses showed a lower cytoplasmatic FABP content and lower HAD and CS activities in type 2 diabetes compared with controls. As far as we are aware, this is the first study that demonstrates a diminished cytoplasmatic FABP content in skeletal muscle of type 2 diabetic subjects. Because FABP functions in the intracellular transport of long-chain fatty acids (11), these data suggest that a lowered cytoplasmatic fatty acid transport may also contribute to the disturbances in muscle fat utilization in type 2 diabetes.
In summary, this study demonstrates that the previously reported
impairment in skeletal muscle fat oxidation in type 2 diabetic subjects
also extends to -adrenergically mediated conditions. Additionally,
it was demonstrated that both the uptake and oxidation of plasma fatty
acids are impaired in skeletal muscle of type 2 diabetic subjects. We
suggest that the mechanisms behind the reduced plasma FFA uptake and
oxidation in skeletal muscle of type 2 diabetic patients may involve
1) the flooding of muscle with FFA generated by muscle
lipolysis, leading to a decrease of the blood-muscle FFA gradient and
2) a decrease of the fatty acid transport capacity as a
consequence of a reduced FABP content.
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ACKNOWLEDGEMENTS |
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We thank Maurice Pelsers and Jos Stegen for analytical support.
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FOOTNOTES |
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This study was supported by a grant from the Dutch Diabetes Research Foundation.
Address for reprint requests and other correspondence: E. E. Blaak, Dept of Human Biology, Nutrition Research Centre, Maastricht Univ., PO Box 616, 6200 MD Maastricht, The Netherlands (E-mail E.Blaak{at}HB.Unimaas.nl).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 30 September 1999; accepted in final form 16 February 2000.
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