Glycogen Synthesis in Human Gastrocnemius Muscle Is

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Glycogen Synthesis in Human Gastrocnemius Muscle Is
Not Representative of Whole-Body Muscle Glycogen
Synthesis
Mireille J.M. Serlie,1 Jacco H. de Haan,2 Cees J. Tack,3 Hein J. Verberne,4 Mariette T. Ackermans,5
Arend Heerschap,2 and Hans P. Sauerwein1
The introduction of 13C magnetic resonance spectroscopy (MRS) has enabled noninvasive measurement of
muscle glycogen synthesis in humans. Conclusions
based on measurements by the MRS technique assume
that glucose metabolism in gastrocnemius muscle is
representative for all skeletal muscles and thus can be
extrapolated to whole-body muscle glucose metabolism.
An alternative method to assess whole-body muscle
glycogen synthesis is the use of [3-3H]glucose. In the
present study, we compared this method to the MRS
technique, which is a well-validated technique for measuring muscle glycogen synthesis. Muscle glycogen synthesis was measured in the gastrocnemius muscle of six
lean healthy subjects by MRS and by the isotope method
during a hyperinsulinemic-euglycemic clamp. Mean muscle glycogen synthesis as measured by the isotope
method was 115 ⴞ 26 ␮mol 䡠 kgⴚ1 muscle 䡠 minⴚ1 vs.
178 ⴞ 72 ␮mol 䡠 kgⴚ1 muscle 䡠 minⴚ1 (P ⴝ 0.03) measured
by MRS. Glycogen synthesis rates measured by MRS
exceeded 100% of glucose uptake in three of the six
subjects. We conclude that glycogen synthesis rates
measured in gastrocnemius muscle cannot be extrapolated to whole-body muscle glycogen synthesis.
Diabetes 54:1277–1282, 2005
D
ecreased insulin sensitivity is a key feature of
type 2 diabetes. The exact pathophysiological
mechanism underlying the defect in insulin
action is still not fully understood. Under conditions of euglycemic hyperinsulinemia, glycogen synthesis accounts for 60 – 80% of glucose disposal in healthy
From the 1Departments of Endocrinology and Metabolism, Academic Medical
Centre, Amsterdam, the Netherlands; the 2Department of Radiology, University Medical Centre Nijmegen, Nijmegen, the Netherlands; the 3Deparment of
General Internal Medicine, University Medical Centre Nijmegen, Nijmegen,
the Netherlands; the 4Department of Nuclear Medicine, Laboratory of Endocrinology and Radiochemistry, Academic Medical Centre, Amsterdam, the
Netherlands; and the 5Department of Clinical Chemistry, Laboratory of
Endocrinology and Radiochemistry, Academic Medical Centre, Amsterdam,
the Netherlands.
Address correspondence and reprint requests to M.J.M. Serlie, Academic
Medical Centre, Department of Endocrinology and Metabolism (F5-169),
Meibergdreef 9, 1105AZ Amsterdam, Netherlands. E-mail: [email protected]
Received for publication 20 April 2004 and accepted in revised form 27
January 2005.
EGP, endogenous glucose production; GIR, glucose infusion rate; IMCL,
intramyocellular lipid concentration; MRS, magnetic resonance spectroscopy;
Rd, rate of glucose disposal.
© 2005 by the American Diabetes Association.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked “advertisement” in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
DIABETES, VOL. 54, MAY 2005
subjects (1), whereas in insulin-resistant states, glycogen
synthesis is decreased (2). Several mechanisms have been
proposed to explain the decreased insulin-induced glycogen synthesis.
With the introduction of 13C magnetic resonance spectroscopy (MRS) by Shulman and colleagues (2– 4), it
became possible to assess glycogen synthesis in humans in
vivo. In 1999, Cline et al. (5) measured glycogen synthesis
with 13C MRS in the gastrocnemius muscle of patients with
type 2 diabetes and healthy control subjects under hyperinsulinemic-hyperglycemic conditions. A decrease by 80%
in glycogen synthesis was found in type 2 diabetic patients
compared with control subjects. Because the intracellular
glucose-6-phosphate concentration in muscle cells (as
measured by 31P MRS) was 1/25 of what would be expected if hexokinase was the rate-limiting step, it was
concluded that the decrease in glycogen synthesis was
caused by an impaired insulin-stimulated glucose transport. Up until now, this is the predominant view on insulin
resistance. Conclusions drawn from 13C MRS measurements are based on the assumption that glucose metabolism in gastrocnemius muscle is representative for muscle
in general and thus can be extrapolated to whole-body
muscle glucose metabolism.
In 1993, Rossetti et al. (6) introduced an alternative
method to measure glycogen synthesis using [3-3H]glucose. During glycolysis, [3-3H]glucose loses its tritium
atom completely to water. Another pathway of tritium loss
may be during fructose-6-phosphate or pentose phosphate
cycling. However, these cycles contribute for a minor part
to glucose turnover (6). No significant label loss or recycling interferes with the results. The appearance of 3H2O in
plasma therefore reflects the rate of whole-body glycolysis. Glycogen synthesis is then calculated by subtracting
glycolysis from the rate of glucose disposal as measured
during a hyperinsulinemic clamp. This method was validated by measuring the incorporation of [3H]glucose in
muscle glycogen in biopsies taken from the vastus lateralis
muscle (6). To our knowledge, the indirect measurement
of glycogen synthesis has never been validated by 13C
MRS, which is considered a well-validated technique for
measuring human gastrocnemius muscle glycogen synthesis in vivo.
The aim of our study was to compare the results of
whole-body muscle glycogen synthesis rates derived from
the extrapolation of direct measured glycogen synthesis
rates in gastrocnemius muscle by 13C MRS with the results
1277
GLYCOGEN SYNTHESIS IN HUMAN GASTROCNEMIUS MUSCLE
of calculation of whole-body muscle glycogen synthesis
rates measured by the isotope method introduced by
Rossetti et al. (6).
RESEARCH DESIGN AND METHODS
We recruited six healthy nonobese young adults (three men and three
women). They were not taking any medication except for oral contraceptives,
had no family history of diabetes, and had a stable weight 3 months before the
study. They did not perform any form of vigorous exercise.
All subjects were studied twice, serving as his or her own control. To rule
out order effects, studies were done in balanced assignment. The 13C MRS
studies were performed in the University Medical Center Nijmegen, Nijmegen,
the Netherlands, and the isotope studies were done in the Academic Medical
Center in Amsterdam. The medical ethical committees of both hospitals
approved the study protocol, and all participants gave written informed
consent.
Measurement of glycogen synthesis by 13C MRS (MRS study). The
subjects attended the University Medical Centre Nijmegen for 13C MRS after
an overnight fast (14 h). Three days before the study, they consumed at least
250 g carbohydrates per day.
Insulin (100 kU Actrapid/l; Novo Nordisk, Alphen aan de Rijn, the Netherlands) infusion was started and continuously infused (60 mU 䡠 m⫺2 body
surface area 䡠 min⫺1) during the experimental protocol for at least 120 min.
For the 13C MRS study, glucose (20% wt/vol) in water was infused to maintain
the plasma glucose concentration at 5 mmol/l. The glucose solution was 30%
enriched at the C-1 position (Campro Scientific, Veenendaal, the Netherlands)
to enrich plasma glucose with [1-13C]glucose. Blood samples were obtained at
7.5-min intervals for measurement of plasma glucose concentration, at 15-min
intervals for fractional plasma glucose 13C enrichment or atom percentage
excess, and at 60-min intervals for plasma insulin. Plasma glucose concentration was measured in duplicate by the glucose oxidase method using a
Beckman Glucose Analyzer II (Beckman, Fullerton, CA).
During the measurements, the subjects were lying inside the magnet of the
magnetic resonance spectrometer (1.5 T Magnetom Vision; Siemens, Erlangen, Germany) with the calf muscle of the right leg positioned on top of a
custom-made radiofrequency coil. For 13C magnetic resonance data acquisition, a concentric surface coil of 13 cm in diameter was used. For 1H
acquisition, decoupling, and shimming, a circularly polarized coil of 2 ⫻ 15 cm
in diameter was used (7). 13C magnetic resonance spectra were obtained in
7.5-min blocks consisting of 2,500 scans using an adiabatic pulse (2,560 ␮s
length) and a repetition time of 180 ms. During the first 60 ms of the
acquisition period, continuous wave decoupling at 26 W was applied, staying
below the specific absorption rate safety limits (8). Increments in muscle
glycogen concentration were calculated from the change in [1-13C]glycogen
integral and the plasma [1-13C]glucose atom percentage excess. The rate of
muscle glycogen synthesis was calculated from the slope of the least-squares
linear fit to the glycogen concentration curve between t ⫽ 30 –120 min and
expressed in micromoles per kilogram muscle per minute.
Measurement of glycogen synthesis by radioisotopes and stable isotopes (isotope study). The subjects followed a diet with at least 250 g
carbohydrates for 3 days before the study. They were admitted to the
metabolic unit of the Academic Medical Centre of the University of Amsterdam and studied in the supine position. At 0900, after the subjects had fasted
overnight (for 14 h), a catheter was inserted into an antecubital vein of each
arm. One catheter was used to sample arterialized blood with use of a heated
hand box (60°C). The other catheter was used to infuse [6,6-2H2]glucose,
[3-3H]glucose, a 20%-glucose solution, and insulin. After a blood sample was
taken to measure the background enrichment and specific activity of plasma
glucose, a primed continuous infusion of [6,6-2H2]glucose (⬎99% enriched;
Cambridge Isotope Laboratories, Cambridge, MA) at a rate of 0.22 ␮mol 䡠 kg⫺1
䡠 min⫺1 (prime: 17.6 ␮mol/kg) and a primed continuous infusion of [3-3H]glucose (74 kBq/ml; Amersham Biosciences, Roosendaal, the Netherlands) at a
rate of 0.0032 ␮Ci 䡠 kg⫺1 䡠 min⫺1 (prime: 0.4 ␮Ci/kg) was started (T ⫽ 0). At the
same time, insulin infusion (100 kU Actrapid/l; Novo Nordisk) was started at
a rate of 60 mU 䡠 m⫺2 body surface area 䡠 min⫺1. After 60 min, blood samples
were drawn for 2 h at 10-min intervals for measurement of specific activity of
plasma glucose and H2O to calculate glycolysis. Between 160 and 200 min, a
blood sample was drawn every 10 min for measurement of isotopic enrichment to calculate rate of glucose disposal. Plasma glucose concentrations
were measured every 5 min with a Beckman Glucose Analyzer II, and the
20%-glucose solution was infused at a variable rate to maintain euglycemia at
5.0 mmol/l. [6,6-2H2]glucose and [3-3H]glucose were added to the 20%-glucose
infusate to approximate the values for enrichment and specific activity
reached in plasma to prevent negative rate of glucose disposal (Rd) artifacts
during the clamp. After 60 and 180 min, blood samples were drawn for
1278
measurement of insulin. During the study, subjects were allowed to drink
water only.
Whole-body composition, appendicular fat, and lean body mass. Wholebody composition was measured with dual-energy X-ray absorptiometry using
a total-body scanner (model QDR 4500 W; Hologic, Waltham, MA). This
scanner produces two X-ray beams at 100 and 140 kVp. After passing through
the body, the attenuated beams are detected by multiple detectors. Attenuation of the two beams depends on mass and type of tissue. Based on regional
attenuation, bone mineral content, total fat mass, total body lean mass (i.e.,
fat-free mass), and lean mass of legs and arms were calculated (9). Appendicular lean mass (i.e., muscle mass in the extremities) was calculated from
the sum of the lean mass of arms and legs (10) after subtraction of bone mass.
Muscle mass in the extremities was assumed to represent total muscle mass.
Total body water was estimated as 60% of total body weight in men and 50%
of total body weight in women (11).
Analytical procedures
Assessment of 13C fractional enrichment. Blood plasma glucose enrichment levels were measured using high-resolution proton nuclear magnetic
resonance (11.7 T). Preparation of plasma samples before nuclear magnetic
resonance consisted of deproteinization by centrifugation for 1 h at 3,000g
over a 10-kDa filter (Sartorius, Göttingen, Germany). From the filtrate, 500 ␮l
was taken, and 20 ␮l D2O with 2,2,3,3-tetradeuteropropionic acid as internal
standard was added. Proton spectra were recorded on an AMX-500 spectrometer (Bruker, Karlsruhe, Germany). The number of averages was 128, and a
repetition time of 10 s was used. Spectra were analyzed using WIN-MR
software (Bruker). Fractional enrichment was calculated from the ratios of
the Lorentzian fitted signals of the proton attached to [12C/13C-1]glucose.
Gas chromatography–mass spectrometry. Plasma samples for glucose
enrichment of [6,6-2H2]glucose were deproteinized with methanol. The aldonitril pentaacetate derivative of glucose was injected into a gas chromatograph–mass spectrometer system (HP 6890 series II gas chromatograph
equipped with a split-splitless injector and an HP 5973 model mass selective
detector; Hewlett-Packard, Palo Alto, CA). Separation was achieved on a
DB17 column (30 m ⫻ 0.25 mm, film thickness of 0.25 ␮m; J&W Scientific,
Folsom, CA). Glucose was monitored at mass-to-charge ratios of 187, 188, and
189. Within each series, three control samples with known enrichments were
measured for quality control. Glucose enrichments were calculated by dividing the area of the mass-to-charge 189 peak by that of the 187 peak (M2:M0)
and correction for natural enrichments.
[3-3H]glucose and 3H2O. Titriated water and [3-3H]glucose were measured
as previously described by Rossetti et al. (6).
Insulin. The plasma insulin concentration was determined by radioimmunoassay (Insulin RIA 100; Pharmacia Diagnostic, Uppsala, Sweden) with an
intra-assay coefficient of variation (CV) of 3–5%, an interassay CV of 6 –9%, and
a detection limit of 15 pmol/l.
Calculations and statistical analysis
Glycogen synthesis by MRS. The rate of glycogen synthesis was calculated
from the slope of the increase of glycogen obtained by linear regression from
increments in glycogen concentration as derived by Shulman et al. (2):
⌬关Gly, t] ⫽ [Agly, t ⫺ Agly, T)/Agly, 0] ⫻ {1.1 ⫻ [Gly0]/FE(t)}
with ⌬[Gly, t] ⫽ glycogen concentration increment at time t. The increment is
calculated from the data point at time T to the next data point at time t. Agly,
t or T or 0 are the resonance areas of the glycogen C-1 signal at t ⫽ t, t ⫽ T, or
t ⫽ 0. [Gly0] ⫽ concentration of glycogen at t ⫽ 0, and FE (t) ⫽ fractional
enrichment at time t.
For the quantification of glycogen ([Gly0]), a phantom containing 100
mmol/l glycogen (rabbit liver glycogen; Sigma, St. Louis, MO), 50 mmol/l
potassium chloride, 40 mmol/l creatinine, and 0.02% sodium azide was used
according to a previously described method (12). Differences in volumes seen
by the 13C coil were determined by integration of the B1 profile of the 13C coil
over the segmented volume of both the entire phantom (Vphantom) and the
skeletal muscle (Vmuscle). Segmentation was performed on T1 weighted
magnetic resonance images acquired with the 1H coil (3D FLASH sequence
[Tr/TE 8.1/4.0 ms], FoV 200 ⫻ 200 mm2, slice thickness 5 mm). Corrections for
differences in coil-loading were determined by using the acquired 13C signal
from a 5-ml reference phantom (10 mmol/l 100% 13C-labeled aceton) at a fixed
position inside the 13C coil. The absolute glycogen content could be estimated
from the following:
[Gly0] ⫽
Amuscle, glycogen ⫻ Aaceton, phantom ⫻ Vphantom ⫻ Glyphantom
Aphantom, glycogen ⫻ Vmuscle ⫻ Aaceton, muscle
with Aglycogen being the glycogen resonance area at 100.5 ppm signal, Aaceton
being the area of the aceton resonance at 200 ppm during muscle or phantom
measurement, Vmuscle being the segmented volume of the muscle area visible
by the coil, and Glyphantom being the glycogen concentration in the phantom.
DIABETES, VOL. 54, MAY 2005
M.J.M. SERLIE AND ASSOCIATES
FIG. 1. Mean GIRs (M value) during the hyperinsulinemic-euglycemic clamp to maintain plasma
glucose at 5 mmol/l. 䉬, M value isotope study; f, M
value MRS study.
The glucose infusion rate (GIR) to maintain euglycemia during the clamp is
expressed as the M value.
Glycogen synthesis by radioisotopes and stable isotopes. When Rd is
calculated, the added source of labeled glucose entering the system and the
exogenous glucose infusate should be taken into account. Thus, Rd was
calculated using Steele equations for non–steady-state conditions adapted for
stable isotopes (13). Reported Rd values represent the mean values from 60 to
120 min in the MRS study and from 160 to 200 min in the isotope study after
the insulin infusion began. Glycolysis was calculated as described by Rossetti
et al. (6). The slope of the linear regression line of plasma 3H2O concentration
in time multiplied by the body water volume estimates the whole-body 3H2O
production rate. Figure 3 shows a representative example of the regression
line of plasma 3H2O concentration in time. The rate of glycolysis was obtained
by dividing the 3H2O production rate by the specific activity of [3-3H]glucose.
Glycogen synthesis was then obtained by subtracting glycolysis from Rd and
dividing glycogen synthesis by the muscle mass, measured by dual-energy
X-ray absorptiometry and multiplying it by body weight.
We used two tracers for our calculations ([3-3H]glucose to measure
glycolysis and [6,6-2H2]glucose to measure Rd) because, in the literature, an
underestimation of glucose turnover is reported because of [3-3H] recycling
(14), and the stable isotope tracer method is too insensitive to measure
enrichment in body water (pilot study; data not shown).
Gas chromatography–mass spectrometry was used in our study to determine isotopic enrichment, and therefore recycled glucose (M⫹1) is excluded
from the tracer-to-tracee ratio measurements, thereby not underestimating
the true flux. All flux rates were expressed as micromoles per kilogram per
minute, except for glycogen synthesis (which was expressed as micromoles
per kilogram muscle per minute). All data are means ⫾ SD.
Statistical analysis was assessed with Student’s t tests, where P ⬍ 0.05 was
defined as statistically significant.
were not different (23 ⫾ 8.8 pmol/l in the isotope study and
27 ⫾ 13.8 pmol/l in the MRS study, P ⫽ 0.63). Furthermore,
the GIRs in both studies were the same, indicating that
there was no endogenous glucose absorption from the gut,
because this would result in much more unpredictable and
unstable GIRs.
The Rd from 160 to 200 min in the isotope study (55 ⫾ 17
␮mol 䡠 kg⫺1 䡠 min⫺1) was equal to the GIR (53.8 ⫾ 15 ␮mol
䡠 kg⫺1 䡠 min⫺1, P ⫽ 0.84), indicating that endogenous
glucose production (EGP) was completely suppressed.
The Rd in the MRS study from 60 to 120 min was 57 ⫾ 18
␮mol 䡠 kg⫺1 䡠 min⫺1 and the M value was 52 ⫾ 17 ␮mol 䡠
kg⫺1 䡠 min⫺1 (P ⫽ 0.04), which indicates an underestimation of the Rd using the M value (from 30 to 60 min) by
⬃8%. However, the GIR (Fig. 1) to maintain euglycemia in
the MRS study did not change from 30 min onward (8.76 ⫾
2.24 mg 䡠 kg⫺1 䡠 min⫺1 from 30 to 60 min, 9 ⫾ 0.5 mg 䡠 kg⫺1
䡠 min⫺1 from 60 to 90 min [P ⫽ 0.58], and 9 ⫾ 2.98 mg 䡠 kg⫺1
䡠 min⫺1 from 90 to 120 min [P ⫽ 0.2 and P ⫽ 0.78,
respectively]), indicating that also between 30 and 60 min
after starting the insulin infusion, EGP contributed maximally 8% to the Rd. The rates of disposal of glucose in the
isotope study and in the MRS study were not significantly
different (55 ⫾ 17 and 57 ⫾ 18 ␮mol 䡠 kg⫺1 䡠 min⫺1,
respectively; Table 2).
RESULTS
We included six healthy young adults. Their characteristics are shown in Table 1. Mean muscle mass was 22.4 ⫾
5.7 kg or 32.8% (range 24 – 42) of body weight. The volunteers were all studied after an overnight fast of 14 h. On
both occasions, they were admitted on the day of the
study. Fasting insulin concentrations were all low, indicating a comparable fasting state on both occasions. The
fasting insulin concentrations in the first and second study
TABLE 1
Characteristics of study participants
Men (n)
Women (n)
Age (years)
Weight (kg)
BMI (kg/m2)
Fat-free mass (kg)
Muscle mass (kg)
Muscle mass (% of weight)
3
3
23.5 (19–31)
67.5 ⫾ 7.7
22.3 ⫾ 1.2
50 ⫾ 9.8
22.4 ⫾ 5.7
32.8 ⫾ 5.8 (24–42)
Data are means ⫾ SD except for age and muscle mass (range).
DIABETES, VOL. 54, MAY 2005
FIG. 2. Glycogen concentration increase (mmol/kg muscle), corrected
for fractional enrichment, as a function of time during the euglycemichyperinsulinemic clamp. Each data point is an average of six individual
data points grouped from measurement times that may vary in time
within 3 min from the indicated time points. The error bars indicate SE.
The straight line is the result of the least-squares fit through the
averaged data points (R2 ⴝ 0.983).
1279
GLYCOGEN SYNTHESIS IN HUMAN GASTROCNEMIUS MUSCLE
TABLE 2
Parameter values of euglycemic-hyperinsulinemic clamp conditions
Isotope
study
MRS
P
Plasma insulin (pmol/l)
588 ⫾ 137 678 ⫾ 124
Plasma glucose (mmol/l)
4.75 ⫾ 0.16 5.0 ⫾ 0.46
Rd (␮mol 䡠 kg⫺1 䡠 min⫺1)
55 ⫾ 17
57 ⫾ 18
M value (␮mol 䡠 kg⫺1 䡠 min⫺1)
53.8 ⫾ 15
52 ⫾ 17
Glycolysis (␮mol 䡠 kg⫺1 䡠 min⫺1)
16 ⫾ 6
—
0.07
NS
NS
NS
—
Data are means ⫾ SD. M value is the glucose infusion rate to
maintain euglycemia.
Mean insulin-induced glycogen synthesis as measured
by stable and radioactive isotopes was 115 ⫾ 26 ␮mol 䡠
kg⫺1 muscle 䡠 min⫺1 (Table 3).
Glycogen synthesis rate was measured by assessing the
glycogen C-1 signal by 13C MRS between 30 and 120 min
after the start of infusion of [13C-1]glucose as described
earlier (2,15). In all subjects, glycogen increased virtually
linearly over this time period, as demonstrated in Fig. 2 for
the average time-dependent glycogen concentration increases. The calculation of the individual glycogen synthesis rates was done from the slope of a linear regression
line through the glycogen concentration data of each
examination. Insulin-stimulated glycogen synthesis in gastrocnemius muscle measured in this way by 13C MRS was
178 ⫾ 72 ␮mol 䡠 kg⫺1 muscle 䡠 min⫺1 (P ⫽ 0.03 vs. isotope
study) (Table 3). The glycogen synthesis rate, expressed as
the percentage of Rd in the isotope study, was 69 ⫾ 7%
(range 58 –78), and in the MRS study, it was 103 ⫾ 39%
(range 42–149) with three subjects exceeding 100% of
glucose uptake (P ⫽ 0.06) (Table 3).
DISCUSSION
This study shows that measurements of insulin-induced
glycogen synthesis rates in human skeletal muscle by
isotope methods or by 13C MRS yield different results. This
conclusion is based on the finding that glycogen synthesis
differed significantly despite comparable whole-body glucose disposal and plasma insulin concentration in the two
studies. Whole-body glycogen synthesis expressed as the
percentage of the rate of glucose disposal and extrapolated from 13C MRS gastrocnemius muscle glycogen synthesis exceeded 100% in three of the six subjects.
TABLE 3
Comparison of glycogen synthesis rates measured by the isotope
and the 13C MRS approach
Glycogen synthesis
Isotope study
Subject
1
2
3
4
5
6
Means ⫾ SD
MRS study
␮mol 䡠 kg⫺1
muscle 䡠 min⫺1
% of
Rd
␮mol 䡠 kg⫺1
muscle 䡠 min⫺1
% of Rd
127
127
74
140
132
92
115 ⫾ 26
78
75
58
64
73
68
69 ⫾ 7
253
214
46
191
204
158
178 ⫾ 72*
137
115
42
90
149
83
103 ⫾ 39
*P ⫽ 0.03 vs. mean glycogen synthesis rate in the isotope study.
1280
FIG. 3. Increase in 3H2O in plasma during infusion of tritiated glucose.
SA, specific activity. R2 ⴝ 0.93.
Glycogen synthesis rate assessed by radioisotopes and
stable isotopes was 69% (range 58 –78) of whole-body
glucose uptake in all subjects.
Studies on the regulation of muscle glycogen synthesis
measured by MRS showed independent influences by
plasma insulin and plasma glucose. The glycogen synthesis rates measured by 13C MRS are comparable with rates
reported in the literature (2,5,15–19).
Glycogen synthesis rate assessed by radioisotope methods was validated by Rossetti et al. (6). Glycogen synthesis
expressed as the percentage of Rd was 69% in our study
and 51% in Rossetti’s study.
This difference can be explained by differences in study
design, with ⬃50% higher insulin concentrations in our
study. After glucose is taken up by skeletal muscle, it can
either be oxidized or converted to glycogen. Glycogen
synthesis under hyperinsulinemic conditions accounts for
60 – 80% of disposed glucose (1). This corresponds well
with our present findings of ⬃70% of Rd. The Rd in the MRS
study was calculated from 60 min onward after achieving
isotopic steady-state. The M value during that period was
⬃8% lower than the calculated Rd, meaning that EGP
contributed by ⬃8% to the Rd. Glycogen synthesis rates
were measured between 30 and 120 min. The GIRs to
maintain euglycemia in the MRS study were stable after 30
min as described in RESULTS. Therefore, we concluded that,
already after 30 min, EGP was almost completely suppressed and that the M value between 30 and 60 min can
be used as a reliable representative of peripheral glucose
uptake. This is in accordance with the literature on this
subject (20). Glycogen synthesis rate during hyperinsulinemia is stable, at least from 30 min to over 120 min
onward, as found in all studies that have applied measurements of glycogen synthesis rate using 13C MRS (2,15,21).
Both fluxes (glycogen synthesis and glycolysis) depend
on glucose uptake, represented in our study by the GIRs
and Rd. The percentage of disposed glucose being oxidized
or stored as glycogen is probably not changing within 2 h
of stable hyperinsulinemic-euglycemic conditions, making
a further increase in glycolysis after 60 min of insulin
infusion unlikely. If there was a further insulin-stimulated
increase in glycolysis after 60 min, the percentage of
glycolysis from Rd would be greater and glycogen synthesis less than our reported 70% of Rd. This would make the
difference between the two experiments even more obvious.
Although muscle mass is an important determinant in
DIABETES, VOL. 54, MAY 2005
M.J.M. SERLIE AND ASSOCIATES
calculation of glycogen synthesis rate, it is mostly estimated and not measured. Sometimes rather low percentages are chosen, resulting in a potential underestimation
of the real flux (2). In our MRS study with glycogen
synthesis expressed as a percentage of whole-body glucose uptake, it exceeded 100% in three of the six subjects
studied. Shulman et al. (2) estimated the muscle mass in
his subjects at 26%. Muscle mass, however, can be quite
variable, even in lean nontrained subjects, as we obtained
an average value of 33% with a range of 24 – 42%. Earlier
studies on body composition showed a muscle mass in
nonobese subjects ranging between 26 and 45% (22,23).
Applying a percentage of muscle mass ⬎26% of body
weight, mean glycogen synthesis rates ⬎100% of wholebody glucose uptake are found (2,16). Our assumption that
extremity skeletal muscle mass represents total muscle
mass may cause an underestimation of total muscle mass.
This means that glycogen synthesis rate in the isotope
study would be lower and in the MRS study would be
higher if total muscle mass was used, leading to a greater
difference between the two methods.
Apparently, depending on the design of the study and
the conditions under which the clamp has been performed, glycogen synthesis rates can differ from 68% to
⬎100% of whole-body glucose uptake. Murphy and Hellerstein (24) earlier addressed this issue while comparing
methods of flux measurements and came to comparable
conclusions. Possible explanations for this finding can be
either related to the MRS technique itself or to the use of
the gastrocnemius muscle as reference muscle. Although
there is some discussion about the full visibility of glycogen using 13C MRS (24), in particular for the liver, several
studies have demonstrated that glycogen in skeletal muscle is fully visible by this method, and a close correlation
was found between glycogen content in human gastrocnemius muscle measured by either MRS or biopsy (25,26).
Assuming that the rates we found measured with 13C MRS
are accurate, the only explanation for the high glycogen
synthesis rate (⬎100% of Rd) is that glycogen synthesis
measured in gastrocnemius muscle is higher than in other
skeletal muscles. If so, it follows that measurements in
gastrocnemius muscle are not representative for glycogen
synthesis rate of body muscle mass in general.
The question now rises by what physiological properties
of the different skeletal muscles glycogen synthesis is
influenced? Skeletal muscles consist of different types of
muscle fibers. Type 1 fibers have a high oxidative capacity
and a higher capillary density than type 2b fibers, which
are characterized by a lower oxidative capacity but higher
glycolytic capacity (27). Sensitivity for insulin also differs
between the fiber types as many different studies indicate
(28 –33).
Therefore, the fact that different muscles differ in fiber
type composition may explain differences in insulin-induced glycogen synthesis rate between different muscle
compartments.
Another potential variable is the positive relationship
between tonicity and GLUT4 protein levels in muscle
(16,34). Gastrocnemius muscle is constantly active while
standing (high tonicity). The difference between the glycogen synthesis rates found in the untrained (75% of
glucose uptake, physiologically possible) and trained
DIABETES, VOL. 54, MAY 2005
(107% of glucose uptake, physiologically impossible) gastrocnemius muscle (16) illustrates the impossibility of
extrapolating the flux rates of an individual muscle group
to whole-body muscle mass with a wide spectrum of
muscle fiber types. Fiber type spectrum of whole-body
muscle is not a constant characteristic but depends on
physical activity and BMI.
A third issue that may affect glycogen synthesis rate is
the intramyocellular lipid concentration (IMCL) in different skeletal muscles, which is thought to influence skeletal
muscle insulin sensitivity. Indeed, an inverse correlation
between IMCL in calf muscle measured with 1H MRS and
peripheral glucose uptake was observed (35). However,
this correlation is probably not the same for all lower leg
muscles.
Our conclusion that muscle groups are not representative for skeletal muscle in general is supported by a recent
report on muscle-type specific lipid metabolism in rats
(36). Neumann-Haefelin et al. (36) reported a muscle-type
specific coping with starvation-induced elevated free fatty
acid levels. IMCL in soleus muscle (i.e., oxidative muscle)
remained constant but increased in tibialis anterior muscle
(i.e., glycolytic muscle).
In conclusion, we found a significantly different glycogen
synthesis rate when measuring with stable and radioactive
isotopes compared with 13C MRS. 13C MRS calculated wholebody glycogen synthesis rates were ⬎100% of glucose uptake
in three of the six subjects. The most likely explanation for
this finding is that glycogen synthesis rate in gastrocnemius
muscle is higher than that in other skeletal muscles, and
therefore measurements in gastrocnemius muscle cannot be
extrapolated to whole-body muscle flux rates. The differences in flux rates between muscle groups may be determined by muscle fiber type, tonicity, and probably also by
IMCL content. The effects of these latter factors on insulin
sensitivity of different skeletal muscles are probably not of
the same magnitude for each muscle. Glycogen synthesis
rate in gastrocnemius muscle is thus not always representative of whole-body muscle insulin sensitivity, and one should
be aware of this problem when evaluating potentially beneficial effects of different interventions on insulin sensitivity.
The beneficial effect could only be true for the muscle under
investigation. Future studies to measure glycogen synthesis
in two different skeletal muscles simultaneously using 13C
MRS are needed.
ACKNOWLEDGMENTS
We thank Gideon Allick for excellent contribution to the
experimental work, Marinette van de Graaf for contribution to the analysis of the MRS data, and An Ruiter
(Laboratory of Endocrinology) for analytical support.
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