Plasma FFA utilization and fatty acid-binding protein content are diminished in type 2 diabetic muscle

Ellen E. Blaak1, Anton J. M. Wagenmakers1, Jan F. C. Glatz2, Bruce H. R. Wolffenbuttel3, Gerrit J. Kemerink4, Chris J. M. Langenberg5, Guido A. K. Heidendal4, and Wim H. M. Saris1

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
SUBJECTS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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 beta -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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
SUBJECTS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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, beta -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.


    SUBJECTS AND METHODS
TOP
ABSTRACT
INTRODUCTION
SUBJECTS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Table 1.   Characteristics of the subjects

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 nonselective beta -agonist isoprenaline (Iso). Protocol 2 was performed to determine the acetate recovery factor, which was used to correct the tracer estimations of plasma palmitate oxidation in protocol 1 for label fixation into products of the tricarboxylic acid cycle and in the bicarbonate pool (21). For protocols 1 and 2, the subjects arrived at the laboratory after an overnight fast (of at least 12 h) at 8:00 AM by car or by bus. They were studied while resting supine on a comfortable bed in a room kept at 23-25°C.

On 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 nonselective beta -agonist Iso [90-150 min, in a dose of 20 ng · kg lean body mass (LBM)-1 · min-1].

ISOTOPE INFUSION. After taking background blood and breath samples (see below), an intravenous priming dose of 0.085 mg/kg NaH13CO3 was given. Next, a constant-rate continuous infusion of [U-13C]palmitate was started (0.011 µmol · kg body wt · -1 · min-1) and continued during the entire period via a calibrated infusion pump (IVAC 560 pump, San Diego, CA). The concentration of palmitate in the infusate was measured for each experiment (see Biochemical Methods) so that the exact infusion rate could be determined. The palmitate tracer (60 mg of the potassium salt of [U-13C]palmitate, 99% enriched; Cambridge Isotope Laboratories, Andover, MA) was dissolved in heated sterile water and was passed through a 0.2-µm filter to 5% warm human serum albumin (Central Blood Bank) to make a 0.670 mM solution [0.668 ± 0.016 (SD) mM].

BREATH, BLOOD, AND URINE SAMPLING. Arterial and deep venous blood samples and a breath sample were obtained before the start of the experiment to determine background isotopic enrichment. At time points 10, 20, 30, 40, 50, 60, 75, and 90 min during the baseline period and at time 100, 110, 120, 135, and 150 min during Iso infusion, breath samples were taken to determine the enrichment of CO2 (13C-to-12C ratio) in expired air. Expired air samples were obtained by having the subjects breath normally for 3 min into a mouthpiece connected to a 6.75-liter mixing chamber, and the sample was collected in a 20-ml vacutainer tube. During the entire experiment, O2 and CO2 exchange were determined with an open-circuit ventilated hood system (Oxycon Beta, Mijnhardt, The Netherlands). Urine was collected overnight and at the end of the Iso infusion to calculate the nonprotein respiratory exchange ratio.

After 60, 75, and 90 min (baseline period) and after 120, 135, and 150 min (Iso infusion), forearm blood flow and arterial and venous concentrations of glucose, lactate, glycerol, O2, FFA, palmitate, and CO2 as well as the 13C-to-12C ratio of palmitate and CO2 were determined.

BLOOD FLOW. Total forearm blood flow (TBF) was measured by venous occlusion plethysmography with a mercury strain gauge (Periflow 0699; Janssen Scientific Instruments), as reported previously (4). To obtain an indication whether TBF was representative of forearm skeletal muscle blood flow (SMBF), an estimation of "pure" SMBF was made by simultaneously determining forearm skin blood flow (SBF), forearm subcutaneous adipose tissue blood flow (ATBF), and forearm composition, as described in detail previously (4).

Protocol 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 wt-1 · 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
TBF<IT>=</IT>amount of muscle<IT>×</IT>SMBF<IT>+</IT>amount of skin

×SBF<IT>+</IT>amount of fat<IT>×</IT>ATBF
where units for the amount of tissue are percent of total forearm area/100; units for blood flow are milliliters per 100 ml tissue per minute.

This equation and the assumptions underlying the estimation of SMBF have been discussed in detail elsewhere (2, 4).

Gas exchange monitoring. The metabolic rate was calculated from the rates of O2 uptake (VO2) and CO2 production (VCO2) according to the equation of Weir (26). Carbohydrate and fat oxidation rates were calculated from VO2 and VCO2 and urinary nitrogen excretion (9).

Tracer calculations. The palmitate flux (rate of appearance or rate of disappearance) was calculated as
palmitate flux (&mgr;mol/min)<IT>=</IT><FR><NU>F</NU><DE>E<SUB>plasma palmitate</SUB><IT>−</IT>E<SUB>bkg</SUB></DE></FR><IT>−</IT>F
where F is the infusion rate of palmitate in micromoles per minute, and Eplasma palmitate - Ebkg is the increase in the 13C-to-12C ratio of plasma palmitate during infusion (compared with background).

The FFA flux was calculated by dividing palmitate flux by the fractional contribution of palmitate to total FFA concentration. Fractional recovery of the acetate label in breath CO2 was calculated as follows
acetate recovery<IT>=</IT><FR><NU>(E<SUB>CO<SUB>2</SUB></SUB><IT>−</IT>E<SUB>bkg</SUB>)<IT>×</IT><A><AC>V</AC><AC>˙</AC></A><SC>co</SC><SUB><IT>2</IT></SUB></NU><DE><IT>2×</IT>F</DE></FR>
where F is the infusion rate of acetate in micromoles per minute; ECO2 - Ebkg is the increase in the 13C-to-12C ratio in expired air during infusion (compared with background); VCO2 is the expired CO2 in micromoles per minute; and the number 2 in the denominator is to correct for the number of 13C molecules in acetate.

The percent of infused [U-13C]palmitate oxidized was calculated as
%infused tracer oxidized<IT>=</IT><FR><NU>(E<SUB>CO<SUB>2</SUB></SUB><IT>−</IT>E<SUB>bkg</SUB>)<IT>×</IT><A><AC>V</AC><AC>˙</AC></A><SC>co</SC><SUB><IT>2</IT></SUB></NU><DE><IT>16×</IT>F<IT>×</IT>acetate recovery</DE></FR>

×100%
where F is the infusion rate of palmitate in micromoles per minute, and the number 16 in the denominator is to correct for the number of 13C molecules in palmitate.

Plasma FFA oxidation (µmol/min) was calculated as
 oxidation<IT>=</IT>FFA flux<IT>×</IT>%infused palmitate tracer oxidized

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 muscle-1 · 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.

The fractional extraction (Fract) of palmitate across the forearm was calculated by dividing the arteriovenous concentration differences of [U-13C]palmitate by the arterial [U-13C]palmitate concentration. Forearm FFA uptake was calculated as follows
forearm muscle FFA uptake<IT>=</IT>fract<IT>×</IT>[FFA<SUB>art</SUB>]<IT>×</IT>PBF
where the units are nanomoles per 100 ml forearm tissue per minute; [FFAart] is arterial FFA concentration; and PBF is forearm plasma blood flow.

Forearm FFA release was calculated from the formula
forearm muscle FFA uptake

 =forearm muscle FFA net balance

−forearm muscle FFA uptake
The oxidation of plasma FFA by skeletal muscle was calculated as
forearm muscle plasma FFA oxidation

 =<FR><NU>TBF<IT>×</IT>(ven<SUP> 13</SUP>CO<SUB>2</SUB> concn<IT>−</IT>art<SUP> 13</SUP>CO<SUB>2</SUB> concn)</NU><DE><IT>16×</IT>[<SUP>13</SUP>C]palmitate uptake<IT>×</IT>acetate recovery</DE></FR>

×muscle FFA uptake
where the units for oxidation and palmitate flux are nanomoles per 100 ml per minute; ven 13CO2 concn is venous ECO2 × venous CO2 concentration; and art 13CO2 concn is arterial 13CO2 concentration.

In this equation, the acetate recovery across forearm muscle is assumed to be similar to the whole body acetate recovery factor (16).

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
TOP
ABSTRACT
INTRODUCTION
SUBJECTS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   Fractional 13C label recovery of [1,2-13C]acetate during baseline (0-90 min) and isoprenaline (Iso)-stimulated conditions (91-150 min) in control and type 2 diabetic subjects. For the Iso effect, P < 0.001; for group effect, P < 0.001; and for interaction of group × Iso effect, P < 0.001.

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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Table 2.   Arterial concentrations of metabolites during baseline conditions and during intravenous infusion of the nonselective beta -agonist Iso

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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Table 3.   Whole body energy expenditure and substrate utilization and plasma FFA flux and oxidation during baseline conditions and during intravenous infusion of the nonselective beta -agonist Iso

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 tissue-1 · 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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Table 4.   TBF, SMBF, and skeletal muscle substrate exchange during baseline conditions and during intravenous infusion of the nonselective beta -agonist Iso

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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Fig. 2.   Forearm skeletal muscle free fatty acid (FFA) uptake, release, and oxidation (in nmol · 100 ml forearm tissue-1 · min-1) during baseline and Iso-stimulated conditions in control and type 2 diabetic subjects. In A and B, P < 0.05 for Iso effect and group effect. In C, P < 0.05 for Iso effect and interaction of group × Iso effect.



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Fig. 3.   Forearm skeletal muscle FFA uptake expressed as percentage of total FFA flux during baseline and Iso-stimulated conditions in control and type 2 diabetic subjects. For group effect: P < 0.05.

During baseline conditions, no significant muscle 13CO2 production and thus no significant oxidation of plasma FFA by muscle could be detected in both groups. During Iso infusion, plasma FFA oxidation by the forearm muscle was significantly increased in controls and still was not significantly different from a zero exchange in type 2 diabetes. A correction for the 13C label fixation by means of the whole body acetate recovery factor yielded an oxidation of plasma FFA accounting for 50% of muscle FFA uptake in controls and no significant oxidation of plasma FFA in type 2 diabetes (Fig. 2).

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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Table 5.   Skeletal muscle cytoplasmatic FABP content and CS, HAD, and LDH activity


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
SUBJECTS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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 beta -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.


    ACKNOWLEDGEMENTS

We thank Maurice Pelsers and Jos Stegen for analytical support.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
SUBJECTS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Bergmeyer, HU. 3-Hydroxyacyl CoA dehydrogenase. Lactate dehydrogenase. In: Methods of Enzymatic Analysis. New York: Academic, 1974, vol. 1, p. 474-476 and 480-482.

2.   Blaak, EE, van Baak MA, Kemerink GJ, Pakbiers MTW, Heidendal GAK, and Saris WHM Beta-adrenergic stimulation of whole body energy expenditure and skeletal muscle metabolism in lean and obese men. Am J Physiol Endocrinol Metab 267: E306-E315, 1994[Abstract/Free Full Text].

3.   Blaak, EE, van Baak MA, Kemerink GJ, Pakbiers MTW, Heidendal GAK, and Saris WHM beta -Adrenergic stimulation of whole body energy expenditure and skeletal muscle metabolism in relation to weight reduction in obese men. Am J Physiol Endocrinol Metab 267: E316-E322, 1994[Abstract/Free Full Text].

4.   Blaak, EE, van Baak MA, Kemerink GJ, Pakbiers MTW, Heidendal GAK, and Saris WHM Total forearm blood flow as indicator of skeletal muscle blood flow: effect of subcutaneous adipose tissue blood flow. Clin Sci (Lond) 87: 559-566, 1994[ISI][Medline].

5.   Colberg, SR, Simoneau J, Thaete FL, and Kelley DE. Skeletal muscle utilization of FFA in women with visceral obesity. J Clin Invest 95: 1846-1853, 1994[ISI].

6.   Dagenais, GR, Tancredi RG, and Zierler KL. Free fatty acid oxidation by forearm muscle at rest, and evidence for an intramuscular lipid pool in the human forearm. J Clin Invest 58: 421-431, 1976[ISI][Medline].

7.   Elia, M, Khan K, Calder G, and Kurpad K. Glycerol exchange across the human forearm assessed by a combination of tracer and arteriovenous exchange techniques. Clin Sci (Lond) 84: 99-104, 1993[ISI][Medline].

8.   Evans, WJ, Phinney SD, and Young VR. Suction applied to a muscle biopsy maximizes sample size. Med Sci Sports Exerc 14: 101-102, 1982[ISI][Medline].

9.   Frayn, KN. Calculation of substrate oxidation rates in vivo from gaseous exchange. J Appl Physiol 55: 628-634, 1983[Abstract/Free Full Text].

10.   Frayn, KN, Williams CM, and Arner P. Are increased plasma non-esterified fatty acid concentrations a risk marker for coronary heart disease and other chronic diseases? Clin Sci (Lond) 90: 243-253, 1996[ISI][Medline].

11.   Glatz, JF, and van der Vusse GJ. Cellular fatty acid binding proteins: their function and physiological significance. Prog Lipid Res 35: 243-282, 1996[ISI][Medline].

12.   Hennes, MMI, Dua A, and Kissebah AH. Effects of free fatty acids and glucose on splanchnic insulin dynamics. Diabetes 46: 57-62, 1997[Abstract].

13.   Jansson, PA, Smith U, and Lönnroth P. Microdialysis assessment of adipose tissue metabolism in postabsorptive obese type 2 diabetes subjects. Eur J Clin Invest 25: 584-589, 1995[ISI][Medline].

14.   Kelley, DE, and Simoneau J. Impaired free fatty acid utilization by skeletal muscle in non-insulin dependent diabetes mellitus. J Clin Invest 94: 2349-2356, 1994[ISI][Medline].

15.   Lewis, GF, Vranic M, Harley P, and Giacca A. Fatty acids mediate the acute extrahepatic effects on hepatic glucose output. Diabetes 46: 111-1119, 1997[ISI].

16.   Mittendorfer, B, Sidossis LS, Walser E, Chinkes DL, and Wolfe RR. Regional acetate kinetics and oxidation in human volunteers. Am J Physiol Endocrinol Metab 274: E978-E983, 1998[Abstract/Free Full Text].

17.   Prentki, M, and Corkey BE. Are beta -cell signalling molecules malonyl-Co-A and cytosolic long-chain acyl-CoA implicated in multiple tissue defects of obesity and NIDDM. Diabetes 45: 273-283, 1996[Abstract].

18.   Saloranta, C, and Groop L. Interactions between glucose and FFA metabolism in man. Diabetes Metab 12: 15-36, 1996[ISI].

19.   Schrauwen, P, van Aggel-Leijssen DPC, van Marken Lichtenbelt WD, van Baak MA, Gijsen AP, and Wagenmakers AJM Validation of the [1,2-13C]acetate recovery factor for correction of [U-13C]palmitate oxidation rates. J Physiol (Lond) 513: 215-223, 1998[Abstract/Free Full Text].

20.   Shepherd, D, and Garland PB. Citrate synthase from the rat liver. Methods Enzymol 13: 11-16, 1969.

21.   Sidossis, LS, Coggan AR, Castaldelli A, and Wolfe RR. A new correction factor for the use in tracer estimations of plasma fatty acid oxidation. Am J Physiol Endocrinol Metab 269: E649-E656, 1995[Abstract/Free Full Text].

22.   Sidossis, LS, Coggan AR, Gastaldelli A, and Wolfe RR. Pathway of free fatty acid oxidation in human subjects. J Clin Invest 95: 278-284, 1995[ISI][Medline].

23.   Siri, WE. The gross composition of the body. Adv Biol Med Physiol 4: 239-280, 1956.

24.   Svedberg, J, Björntorp P, Smith U, and Lönnroth P. Free-fatty acid inhibition of insulin binding, degradation, and action in isolated rat hepatocytes. Diabetes 39: 570-574, 1990[Abstract].

25.   Van der Vusse, GJ, and Reneman RS. Lipid metabolism in muscle. In: Handbook of Physiology. Exercise: Regulation and Integration of Multiple Systems. Bethesda, MD: Am Physiol Soc, 1996, sect. 12, chapt. 21, p. 952-994.

26.   Weir, JB. New methods for calculating metabolic rate with special reference to protein metabolism. J Physiol (Lond) 109: 1-9, 1949[ISI].

27.   Wodzig, KWH, Pelsers MMAL, van der Vusse GJ, Roos W, and Glatz JFC One-step enzyme linked immunosorbent assay (ELISA) for plasma fatty acid-binding protein. Ann Clin Biochem 34: 263-268, 1997[ISI][Medline].


Am J Physiol Endocrinol Metab 279(1):E146-E154
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