The Fate of U-13C Palmitate Extracted by Skeletal Muscle in Subjects With Type 2 Diabetes and Control Subjects
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The Fate of [U-13C]Palmitate Extracted by Skeletal
Muscle in Subjects With Type 2 Diabetes and
Control Subjects
Ellen E. Blaak and Anton J.M. Wagenmakers
The current study investigated the fate of a [U-13C]
palmitate tracer extracted by forearm muscle in type 2
T
diabetic and control subjects. We studied seven healthy he 13C-labeled fatty acid tracers are used to
lean male subjects and seven obese male subjects with quantify the oxidation of plasma fatty acids in
type 2 diabetes using the forearm muscle balance tech- several metabolic conditions. However, the re-
nique with continuous intravenous infusion of the sta- covery of 13C-labeled products in expired CO2
ble isotope tracer [U-13C]palmitate under baseline during infusion of 14C- or 13C-acetate is only 20 –30% after
conditions and during intravenous infusion of the non- a 2-h infusion. Sidossis and colleagues (1,2), therefore,
selective -agonist isoprenaline (ISO; 20 ng 䡠 kgⴚ1 lean
body mass 䡠 minⴚ1). In skeletal muscle of control sub-
suggested that the label fixation observed during infusion
jects, there was a significant release of 13C-labeled of a fatty acid tracer occurs mainly via isotopic exchange
oxidation products in the form of 13CO2 (15% of 13C reactions in the tricarboxylic acid (TCA) cycle. The recov-
uptake from labeled palmitate) and a significant release ery of labeled products during infusion of labeled acetate
of 13C-labeled glutamine (release of 13C-labeled atoms (acetate recovery factor) has been proposed to be a good
from glutamine was 6% of 13C uptake from labeled correction factor for tracer estimations of fatty acid oxi-
palmitate), whereas in type 2 diabetic subjects there dation, as it accounts for label fixation that might occur at
was no detectable release of 13CO2 and 13C-glutamine, any step from the entrance of labeled acetyl-CoA into the
despite a significant uptake of [U-13C]palmitate (60% of TCA cycle until the recovery of the labeled product in
control value). There was net uptake of arterial 13C- expired breath CO2. The TCA cycle products in which
labeled glutamate by forearm muscle in both groups.
Also, the ISO-induced increase in arterial glutamine
labeled products from 13C-labeled tracer may accumulate
enrichment and arterial concentration of 13C-glutamine are glutamine, released mainly by skeletal muscle and liver
was more pronounced in the diabetic group relative to (3,4); glutamate, released by gut and liver; and glucose,
control subjects. In view of the diminished ISO-induced formed in the liver by means of gluconeogenesis (4). It has
release of 13C-glutamine from type 2 diabetic muscle, been estimated that nonoxidative 13C loss during a 2-h
the latter data indicate that more [U-13C]palmitate en- [1,2-13C]acetate infusion to glutamate, glutamine, or glu-
tered the liver in the diabetic group and was incorpo- cose amounts to 10, 12, and 0.3%, respectively (5). So far
rated into newly synthesized glutamine and glutamate no information is available on the exchange of 13C-labeled
molecules. Thus, the lack of release of 13C-labeled products across skeletal muscle during infusion of a
oxidation products by type 2 diabetic muscle during 13
C-labeled fatty acid tracer.
-adrenergic stimulation, despite significant [U-13C]-
palmitate uptake, indicates differences in the handling
In type 2 diabetic subjects, the uptake and/or oxidation
of fatty acids between type 2 diabetic subjects and of plasma free fatty acids (FFAs) has been shown to be
healthy control subjects. Diabetes 51:784 –789, 2002 impaired during postabsorptive conditions (6,7), -adren-
ergic stimulation (7), and exercise (8). It has also been
shown that palmitate and acetate 13C-label recovery in
expired breath are lower in type 2 diabetic subjects versus
healthy control subjects (7–9). On the basis of these
findings, we hypothesized that the fate of 13C-labeled fatty
acid tracer extracted by skeletal muscle may differ in type
2 diabetic subjects as compared with healthy volunteers.
The aims of the present study were as follows: 1) establish
the uptake and release of 13C-labeled substrate and oxida-
tion products by skeletal muscle during [U-13C]palmitate
From the Department of Human Biology, Nutrition Research Center, Maas-
infusion under baseline conditions and during infusion of
tricht University, Maastricht, the Netherlands. the nonselective -agonist isoprenaline (ISO) and 2) com-
Address correspondence and reprint requests to Dr. E.E. Blaak, Dept. of pare 13C-label fixation in glutamate, glutamine, and glu-
Human Biology, Nutrition Research Centre, Maastricht University, P.O. Box
616, 6200 MD Maastricht, The Netherlands. E-mail: e.blaak@hb.unimaas.nl. cose in control and type 2 diabetic subjects.
Received for publication 1 May 2001 and accepted in revised form 6
December 2001.
CV, coefficient of variation; FFA, free fatty acid; GC, gas chromatography;
RESEARCH DESIGN AND METHODS
IRMS, isotope ratio mass spectrometer; ISO, isoprenaline; TCA, tricarboxylic Subjects. We studied seven healthy lean male subjects and seven obese male
acid. subjects with type 2 diabetes (diabetes duration 2 years, range 0.5– 8 years).
784 DIABETES, VOL. 51, MARCH 2002E.E. BLAAK AND A.J.M. WAGENMAKERS
TABLE 1 Blood flow. Total forearm blood flow was measured by venous occlusion
Subject characteristics plethysmography with a mercury strain gauge (Periflow 0699; Janssen Scien-
tific Instruments, Belgium), as reported previously (11).
Control Type 2 diabetes Biochemical methods. Blood samples were taken simultaneously from the
radial artery and deep forearm vein after the blood flow measurement while
n 7 7 the hand circulation was still occluded. Duplicate 1-ml blood samples were
Age (years) 50.9 ⫾ 2.1 49.0 ⫾ 2.7 immediately injected with a needle through the rubber stopper of preweighed
Weight (kg) 74.6 ⫾ 2.8 98.7 ⫾ 5.4 vacutainer tubes, without disturbing the vacuum, for the determination of
13
BMI (kg/m2) 24.0 ⫾ 0.8 31.1 ⫾ 3.2 CO2/12CO2. After being weighed again, 1 vol of 1 mol/l H2SO4 was injected
through the rubber stopper into the tubes to direct all blood CO2 into the
Percent body fat 15.1 ⫾ 1.9 32.5 ⫾ 2.2
gaseous head space. The tubes were weighed again to determine the dilution
Waist-to-hip ratio 0.93 ⫾ 0.01 1.05 ⫾ 0.02 factor. The gaseous head space was finally brought to atmospheric pressure
Fasting blood glucose (mmol/l) 5.36 ⫾ 0.21 7.00 ⫾ 0.74 with helium. The same procedure was applied to bicarbonate standards of
known concentration. The coefficient of variation (CV) for this method with
Data are means ⫾ SE.
CO2 concentrations in the 15–30 mmol/l range is 0.4%. The CV between
duplicate measurements of CO2 concentrations in blood is 0.09%.
Data on skeletal muscle fatty acid utilization in these subjects have been All other blood samples were collected in tubes containing EDTA kept on
previously published (7). The diabetic subjects were treated with diet alone ice. These samples were immediately centrifuged at 4°C, and the plasma was
(n ⫽ 2) or diet in combination with oral blood glucose⫺lowering agents (low put in liquid nitrogen until storage at ⫺80°C.
dosages of sulfonylureas, which were withheld for 2 days before the experi- Breath and blood samples were analyzed for their 13C/12C ratio and CO2
ments; n ⫽ 5). Beside this, no other medications were used. None of the content by injecting 20 l of the gaseous head space into a gas chromatogra-
subjects had a serious health problem apart from diabetes. Normal resting phy (GC) continuous flow isotope ratio mass spectrometer (IRMS; Finnigan
electrocardiogram and blood pressure were prerequisites for participation. MAT 252, Bremen, Germany).
Subject characteristics are given in Table 1. All subjects engaged in sports ⱕ3 For the determination of plasma palmitate, FFAs were extracted from
h per week, and none had a physically demanding job. The study protocol was plasma, isolated by thin-layer chromatography, and derivatized to their methyl
approved by the Medical Ethical Review Committee of Maastricht University, esters. Isotope enrichment of palmitate was determined by GC-IRMS after
and all subjects gave written informed consent. on-line combustion of the fatty acids to CO2. The methyl ester of palmitate
Body weight was determined on an electronic scale, accurate to 0.1 kg. contains 17 carbon atoms; therefore, the tracer/tracee ratio of palmitate was
Waist and hip circumference measurements to the nearest 1 cm were made corrected for the extra methyl group.
with the subject standing upright. Body composition was determined by Palmitate concentrations were determined on an analytical GC with
hydrostatic weighing with simultaneous lung volume measurement (Volu- ion-flame detection using heptadecanoic acid as the internal standard. On
graph 2000; Mijnhardt, Bunnı́k, the Netherlands). Body composition was average, it comprised 24.5 ⫾ 0.6% of the total FFA concentration. Plasma
calculated according to Siri’s method (10). glucose, glutamine, and glutamate concentrations (deproteinized with 3.5
Experimental design. The present study was performed to determine wt/vol % sulphosalicyclic acid [SSA]) were measured using standard enzy-
skeletal muscle 13C-palmitate kinetics during baseline and intravenous infu- matic techniques automated (Cobas Fara centrifugal analyzer) at 340 nm.
sion of the nonselective -agonist ISO. Subjects arrived at the laboratory at Isotopic enrichment of plasma glutamine and glutamate was determined as
8:00 A.M. by car or by bus after an overnight fast (at least 12 h). They were the MTBSTFA derivative using GC combustion IRMS (MAT 252; Thermo
studied while resting supine on a comfortable bed in a room kept at 23–25°C. Finnigan, Bremen, Germany). The resulting derivative contained 23 carbon
Forearm skeletal muscle metabolism was studied under baseline condi- atoms, 5 of which were from glutamine or glutamate; the 13C/12C ratio was
tions and during intravenous infusion of ISO by means of the forearm muscle therefore corrected by a factor of 23/5. To determine plasma glucose
balance technique with continuous intravenous infusion of the stable isotope enrichment, glucose was extracted with chloroform-methanol-water, and
tracer [U-13C]palmitate. Before the start of the experiment, three cannulas derivatization occurred with butylboronic acid and acetic anhydride, as
were inserted, as follows: one cannula was inserted under local anesthesia in previously described (12). The resulting derivative contained 16 carbon atoms,
the radial artery of the forearm for sampling of arterial blood; in the same arm, 6 of which were from glucose; the 13C/12C ratio was therefore corrected by a
a second cannula was inserted in a forearm vein for the infusion of ISO and factor of 16/6. The glucose derivatives were analyzed by GC combustion IRMS.
the stable isotope tracer; in the contralateral arm, a third catheter was Calculations
inserted in the retrograde direction in an antecubital vein for the sampling of Tracer calculations. Fractional recovery of the palmitate label in breath
deep venous blood, draining from forearm muscle. Measurements were done CO2 was calculated as follows:
during the last 30 min of a 90-min baseline period (0 –90 min) and a subsequent
60-min period of intravenous infusion of ISO (90 –150 min) given at 20 ng 䡠 (TTRCO2 ⫺ TTRbkg) * VCO2
kg⫺1 lean body mass 䡠 min⫺1. palmitate recovery ⫽
16 * F
Isotope infusion. After taking background blood and breath samples (see
below), an intravenous priming dose of 0.085 mg/kg NaH13CO3 was given. where F is the infusion rate of palmitate in micromoles per minute, TTRCO2 ⫺
Then a constant-rate continuous infusion of [U-13C]palmitate was begun TTRbkg equals the increase in tracer/tracee (13C/12C) ratio in expired air during
(0.011 mol 䡠 kg⫺1 body wt 䡠 min⫺1) and continued during the entire period via infusion (compared with background), and VCO2 is the expired CO2 in
a calibrated infusion pump (IVAC 560 pump; IVAC, San Diego, CA). The micromoles per minute. The number 16 in the denominator is to correct for
concentration of palmitate in the infusate was measured for each experiment the number of 13C atoms in palmitate.
(see BIOCHEMICAL METHODS, below) so that the exact infusion rate could be Forearm muscle calculations. The exchange of metabolites or tracer-
determined. The palmitate tracer (60 mg of the potassium salt of [U-13C]palmi- labeled metabolites across forearm muscle was calculated by multiplying the
tate, 99% enriched; Cambridge Isotope Laboratories, Andover, MA) was arteriovenous concentration difference of metabolites (in micromoles per
dissolved in heated sterile water and passed through a 0.2-m filter into 5% liter) or 13C-labeled atoms in metabolites (13C/12C ratio ⫻ number of C-atoms
warm human serum albumin (Central Blood Bank, the Netherlands) to make in molecule ⫻ metabolite concentration) by forearm plasma flow (ml 䡠 100
a 0.670 mmol/l solution (mean ⫾ SD, 0.668 ⫾ 0.016 mmol/l). ml⫺1 forearm muscle 䡠 min⫺1) or by total forearm blood flow (for CO2
Blood and breath sampling. Arterial and deep venous blood samples and exchange). Forearm plasma flow was calculated by multiplying forearm blood
a breath sample were obtained before the start of the experiment to determine flow with (1⫺hematocrit)/100. A positive exchange indicates uptake.
background isotopic enrichment. Expired-air samples were obtained by Statistical analysis. Data are expressed as means ⫾ SE, unless otherwise
having the subjects breath normally for 3 min into a mouthpiece connected to indicated. To compare baseline and isoprenaline-induced responses between
a 6.75-l mixing chamber and then collecting a sample into a 20-ml vacutainer groups, a two-factor repeated measures ANOVA was performed. P ⬍ 0.05 was
tube. At time points 10, 20, 30, 40, 50, 60, 75, and 90 min during the baseline regarded as statistically significant.
period and 110, 120, 130, 145, and 160 min during ISO infusion, breath samples
were taken to determine the enrichment of CO2 (13C/12C ratio) in expired air.
During the entire experiment, CO2 exchange was determined with an open- RESULTS
circuit ventilated hood system (Oxycon Beta, Jaeger, Breda, the Netherlands). Palmitate recovery. The fractional recovery of
After the 60, 75, and 90 min of the baseline period, and 30, 45, and 60 min
of ISO infusion, forearm blood flow, arterial and venous concentrations, and
[U-13C]palmitate tracer in expired 13CO2 was lower in type
13
C/12C ratios of glucose, palmitate, glutamine, glutamate, and CO2 were 2 diabetic subjects versus control subjects during both
determined. baseline conditions and ISO infusion (P ⬍ 0.001) (Fig. 1).
DIABETES, VOL. 51, MARCH 2002 785TYPE 2 DIABETES AND FATTY ACID HANDLING
FIG. 1. Fractional label recovery of 13C in expired air during
[U-13C]palmitate infusion in control (䡺) and type 2 diabetic subjects
(䉫) during baseline conditions (0 –90 min) and infusion of the nonse-
lective -agonist ISO (91–150 min).
Arterial concentrations. Arterial palmitate concentra-
tions were not significantly different between the two
groups (Table 2), whereas plasma palmitate enrichment
(13C/12C ratio) was higher in type 2 diabetic subjects (Fig. FIG. 2. Arterial plasma palmitate, glutamine, glutamate, and glucose
2). Arterial glucose concentrations were higher in type 2 enrichment (13C/12C ratios) during [U-13C]palmitate infusion in control
(䡺) and type 2 diabetic subjects (䉫) during baseline conditions (60, 90
diabetic subjects and more decreased during ISO infusion min) and infusion of the nonselective -agonist (120, 150 min). E-0.5,
in this group (P ⬍ 0.01). Arterial glucose enrichment (Fig. ⴛ 10ⴚ5.
2) and the concentration of 13C-glucose (defined as 13C/12C
ratio glucose ⫻ glucose concentration ⫻ number of C- groups during baseline conditions or ISO infusion, al-
atoms in glucose) increased throughout the experiment though glutamate concentrations tended to be higher in
(P ⬍ 0.001) and were not significantly different between type 2 diabetic subjects (P ⫽ 0.12). In both groups,
groups. Glutamine concentration tended to be lower in the glutamate concentrations significantly decreased during
type 2 diabetic group (P ⫽ 0.10). Glutamine enrichment ISO infusion (P ⬍ 0.001) (Table 2). Glutamate enrichment
(Fig. 2) and the 13C-glutamine concentration (defined as increased throughout the experiment and was not signifi-
C/ C ratio glutamine ⫻ glutamine concentration ⫻
13 12
cantly different between groups (Fig. 2). The concentra-
number of C-atoms in glutamine) increased significantly tion of 13C-glutamate (defined as 13C/12C ratio glutamate ⫻
throughout the experiment (P ⬍ 0.001); the ISO-induced glutamate concentration ⫻ number of C-atoms in gluta-
increase in glutamine enrichment and 13C-glutamine was mate) tended to be higher in type 2 diabetic subjects than
significantly more pronounced in type 2 diabetic subjects in control subjects (P ⫽ 0.08).
than in control subjects (P ⬍ 0.01). Glutamate and CO2 Skeletal muscle substrate fluxes. Forearm plasma
concentrations were not significantly different between blood flow tended to be lower in type 2 diabetic than in
TABLE 2
Arterial concentrations of metabolites during baseline conditions and intravenous infusion of the nonselective -agonist ISO
Isoprenaline P (ANOVA)
Baseline 120 150 Group ISO Interaction
Palmitate (mol/l)
Control 177 ⫾ 20 276 ⫾ 19 294 ⫾ 19 — 0.001 —
Type 2 155 ⫾ 14 263 ⫾ 31 274 ⫾ 22 — — —
Glucose (mmol/l)
Control 5.36 ⫾ 0.21 5.43 ⫾ 0.18 5.41 ⫾ 0.18 0.10 0.05 0.01
Type 2 7.00 ⫾ 0.74 6.66 ⫾ 0.67 6.55 ⫾ 0.64 — — —
Glutamine (mol/l)
Control 585 ⫾ 16 612 ⫾ 34 612 ⫾ 28 0.10 — 0.11
Type 2 539 ⫾ 42 526 ⫾ 44 509 ⫾ 38 — — —
Glutamate (mol/l)
Control 160 ⫾ 9 121 ⫾ 13 118 ⫾ 5 0.12 0.001 —
Type 2 195 ⫾ 22 157 ⫾ 19 153 ⫾ 23 — — —
CO2 (mmol/l)
Control 23.5 ⫾ 0.8 21.9 ⫾ 0.5 22.6 ⫾ 0.6 — — —
Type 2 22.4 ⫾ 0.7 21.7 ⫾ 0.7 21.3 ⫾ 0.7 — — —
Data are means ⫾ SE or n.
786 DIABETES, VOL. 51, MARCH 2002E.E. BLAAK AND A.J.M. WAGENMAKERS
TABLE 3
13 13
Skeletal muscle fluxes of C-labeled metabolites and CO2 during baseline conditions and intravenous infusion of isoprenaline
P (ANOVA)
Baseline Isoprenaline Group ISO Interaction
Plasma blood flow
Con 1.51 ⫾ 0.22 2.07 ⫾ 0.42 — — —
Type 2 1.15 ⫾ 0.10 1.63 ⫾ 0.12 — — —
Palmitate
Control 15 ⫾ 12 52 ⫾ 34 — — —
Type 2 6⫾7 7⫾9 — — —
13
C-palmitate
Control 6.97 ⫾ 0.84 6.78 ⫾ 0.88 0.06 — —
Type 2 5.01 ⫾ 0.50 4.52 ⫾ 0.75 — — —
Glutamine
Control ⫺185 ⫾ 17 ⫺246 ⫾ 79 — — —
Type 2 ⫺177 ⫾ 31 ⫺175 ⫾ 27 — — —
13
C-glutamine
Control 0.175 ⫾ 0.070 ⫺0.430 ⫾ 0.300 0.10 — 0.05
Type 2 0.095 ⫾ 0.050 0.225 ⫾ 0.140 — — —
Glutamate
Control 100 ⫾ 14 117 ⫾ 39 — — —
Type 2 106 ⫾ 19 122 ⫾ 16 — — —
13
C-glutamate
Control 0.135 ⫾ 0.025 0.265 ⫾ 0.075 — 0.01 —
Type 2 0.150 ⫾ 0.03 0.290 ⫾ 0.045 — — —
Glucose
Control 295 ⫾ 81 268 ⫾ 179 0.08 — —
Type 2 173 ⫾ 42 17 ⫾ 54 — — —
13
C-glucose
Control ⫺0.114 ⫾ 0.186 0.000 ⫾ 0.000 — — —
Type 2 0.153 ⫾ 0.281 0.000 ⫾ 0.000 — — —
CO2
Control ⫺7.94 ⫾ 0.74 ⫺9.06 ⫾ 1.9 0.07 — —
Type 2 ⫺5.54 ⫾ 0.83 ⫺5.96 ⫾ 0.96 — — —
13
CO2
Control ⫺0.02 ⫾ 0.04 ⫺1.02 ⫾ 0.038 0.07 — 0.05
Type 2 0.40 ⫾ 0.013 0.02 ⫾ 0.015 — — —
Data are means ⫾ SE or n. Flux of 13C-labeled metabolites (in nmol 䡠 100 ml⫺1 tissue 䡠 min⫺1) is defined as plasma blood flow ⫻ [(arterial
C/ C ratio ⫻ concentration ⫻ number of C-atoms) ⫺ (venous 13C/12C ratio ⫻ concentration ⫻ number of C-atoms)], with plasma blood
13 12
flow in ml 䡠 100 ml⫺1 tissue 䡠 min⫺1 and concentration in mol/l; see CALCULATIONS.
control subjects (P ⫽ 0.095) (Table 3). Net muscle palmi- skeletal during ISO infusion. In both groups there was a
tate uptake was not significantly different between groups significant uptake of 13C-palmitate (slightly lower in type 2
during baseline or ISO-stimulated conditions. The uptake diabetic subjects; P ⫽ 0.06) and 13C-glutamate. In control
of 13C-palmitate did not change as a result of ISO stimu-
lation and tended to be lower in type 2 diabetic than in
control subjects (P ⫽ 0.06). Glucose uptake tended to be
lower in type 2 diabetic versus control subjects (P ⫽ 0.08),
whereas there were no significant differences in the uptake
of 13C-glucose across muscle between the two groups.
Glutamine release was comparable in both groups during
baseline conditions and ISO infusion. There was a signif-
icant release of 13C-glutamine in control subjects during
ISO infusion, whereas no release could be detected in type
2 diabetic subjects (interaction group ⫻ ISO; P ⬍ 0.05).
Muscle CO2 release did not differ between groups during
baseline conditions or ISO infusion, although values
tended to be lower in type 2 diabetic subjects compared
with control subjects (P ⫽ 0.07). There was significant FIG. 3. 13C carbon balance across skeletal muscle during [U-13C]palmi-
tate infusion in control and type 2 diabetic subjects during infusion of
muscle 13CO2 production during ISO stimulation in control the nonselective -agonist ISO. Flux of 13C-labeled metabolites (in
subjects, whereas we could not detect significant 13CO2 nmol 䡠 100 mlⴚ1 tissue 䡠 minⴚ1) is defined as plasma blood flow ⴛ
release in type 2 diabetic subjects (interaction group ⫻ ([arterial 13C/12C ratio ⴛ concentration ⴛ number of C-atoms] ⴚ
[venous 13C/12C ratio ⴛ concentration ⴛ number of C-atoms]), with
ISO; P ⬍ 0.05). plasma blood flow in ml 䡠 100 mlⴚ1 tissue 䡠 minⴚ1 and with concentration
Figure 3 shows the 13C-labeled carbon balance across in mol/l; see RESEARCH DESIGN AND METHODS.
DIABETES, VOL. 51, MARCH 2002 787TYPE 2 DIABETES AND FATTY ACID HANDLING
subjects, there was significant 13CO2 release (15% of 13C palmitate taken up by muscle was not converted to
uptake from labeled palmitate) and a significant 13C- oxidation products in type 2 diabetic muscle. Then, the
glutamine release (release of 13C-labeled atoms from glu- most likely alternative fate of palmitate in these subjects
tamine was 6% of 13C uptake from labeled palmitate; P ⬍ was synthesis of triglycerides and incorporation in the
0.05). In contrast, in type 2 diabetic subjects, there was no muscle triglyceride stores. On the other hand, we previ-
release of 13C-labeled oxidation products. ously found an increased glycerol release in these subjects
during both baseline conditions and ISO infusion (7),
indicating an increased lipolysis of the triacylglycerol
DISCUSSION
stores and thus possibly an increased intramuscular FFA
The present study investigated the fate of [U-13C]palmitate pool. Therefore, it is also possible that the [U-13C]palmi-
extracted by forearm muscle in type 2 diabetic and control tate tracer was diluted to a larger extent in the intramus-
subjects. In skeletal muscle of control subjects, there was cular FFA pool in type 2 diabetic subjects as compared
a significant release of oxidation products from 13C-palmi- with control subjects, which may have made it more
tate in the form of 13CO2 (15% of 13C uptake from labeled difficult to detect a significant 13C label fixation into
palmitate) and 13C- glutamine (release of 13C atoms from oxidation products. Nevertheless, both explanations imply
glutamine was 6% of 13C uptake from labeled palmitate). In that there are differences in how type 2 diabetic and
contrast, in type 2 diabetic subjects, there was no detect- healthy control subjects handle fatty acids.
able release of 13CO2 and 13C-glutamine, despite a signifi-
Interestingly, the ISO-induced increase in arterial glu-
cant 13C label uptake from [U-13C]palmitate (60% of
tamine enrichment and arterial 13C-glutamine was more
control value). These data indicate differences in FFA
pronounced in the diabetic group. In view of the reduced
handling in skeletal muscle of control and type 2 diabetic 13
subjects during infusion of the nonselective -agonist ISO. C-glutamine production by skeletal muscle in type 2
Baseline conditions. Pathways for label fixation are the diabetic subjects, these data indicate that tissues other
loss of 13C label to glutamine and glutamate or glucose. than skeletal muscle were responsible for the more pro-
Indeed, we found a significant increase in arterial plasma nounced increase in glutamine enrichment during ISO
glutamine, glutamate, and glucose enrichment throughout infusion. As discussed above, the most likely candidate for
the experiment, as previously reported (5). During base- this release of 13C-glutamine is the liver, because the liver
line conditions, no significant release of 13C-glutamine can both take up and release glutamine (high glutaminase
from muscle could be detected in either group, despite a and glutamine synthetase activity) (13,14) and also plays a
significant increase in glutamine enrichment and 13C- central role in fatty acid metabolism. The higher increase
glutamine in arterial blood. These data indicate that there in arterial glutamine enrichment in type 2 diabetic subjects
was a more rapid release of 13C-glutamine by other tissues as compared with control subjects indicated that more
than by skeletal muscle. It has been previously reported [U-13C]palmitate entered the liver in the former group,
that the liver can both take up and release glutamine by where more 13C label accumulated in glutamine. Support
the periportal parenchymal (at inflow site) and perivenous for this idea also comes from the finding that the concen-
cells, respectively. In the perivenous liver cells (situated at tration of 13C-glutamate tends to be higher in arterial blood
liver outflow), a sodium-dependent glutamate transporter of type 2 diabetic subjects, a finding most likely explained
(responsible for glutamate uptake against a concentration by an increased incorporation of 13C label from palmitate
gradient) and glutamine synthetase are exclusively ex- into glutamate in the liver. An increased FFA supply to the
pressed (13,14). For this reason, perivenous cells react liver may promote hepatic gluconeogenesis and glucose
similarly to muscle; they extract glutamate (from circula- output (15), decrease insulin binding to hepatocytes (16),
tion or formed in the TCA cycle) and use it for glutamine and promote VLDL triglyceride production (17), all impor-
synthesis. Because of the central role of the liver in tant factors in the etiology of insulin resistance, type 2
glutamine/glutamate homeostasis and fatty acid metabo- diabetes, and cardiovascular disease.
lism, the liver seems to be the most likely site for the initial Thus the release of 13C-labeled oxidation products dur-
increase in arterial glutamine, glutamate, and glucose ing [U-13C]palmitate infusion was lower in muscle of type
enrichments. 2 diabetic subjects than in control subjects. Mittendorfer
During baseline conditions, there was no significant et al. (18) reported that the 13C label recovery during
13
CO2 production by skeletal muscle in either group, [1,2-13C]acetate infusion was similar across muscle, across
indicating there was no oxidation of plasma palmitate. the splanchnic bed, and at the whole-body level in healthy
However, we cannot exclude the possibility that 13CO2 subjects. In view of the differences in the exchange of
13
production could not be measured with the present infu- C-substrates and products across muscle in type 2 dia-
sion rate, as the recovery of 13CO2 from a palmitate tracer betic subjects relative to control subjects, it would be
was low (7% recovery in expired CO2 after 90-min infu- worthwhile to study whether the above assumption also
sion) (Fig. 1) and increased linearly in time for periods of holds for type 2 diabetic subjects.
10 –11 h. This point has been previously discussed (7). In summary, in muscle of type 2 diabetic subjects, there
Isoprenaline stimulation. As indicated above, in type 2 was no detectable release of 13C label into oxidation
diabetic subjects there was no detectable release of 13CO2 products during ISO infusion, despite a significant uptake
and 13C-glutamine despite a significant uptake of 13C label of 13C label from [U-13C]palmitate; in contrast, in control
from [U-13C]palmitate (60% of control value), whereas in subjects, there was a significant release of 13CO2 and
control subjects there was a significant release of 13CO2 13
C-glutamine. We propose the following explanations for
and 13C-glutamine. Several mechanisms may be responsi- this finding: First, the lack of release of 13C-oxidation
ble for this finding. First, these data may indicate that the products may indicate that more palmitate was incorpo-
788 DIABETES, VOL. 51, MARCH 2002E.E. BLAAK AND A.J.M. WAGENMAKERS
rated in the triglyceride stores in type 2 diabetic muscle. skeletal muscle of type 2 diabetic subjects. Am J Physiol 279:E146 –E154,
Because an increased intramuscular triglyceride content is 2000
8. Blaak EE, van Aggel-Leijssen DPC, Wagenmakers AJM, Saris WHM, van
strongly linked to insulin resistance in lean offspring of Baak MA: Impaired oxidation of plasma-derived fatty acids in type 2
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