Download Addition of protein and amino acids to carbohydrates

J Appl Physiol
91: 839–846, 2001.
Addition of protein and amino acids to carbohydrates
does not enhance postexercise muscle glycogen synthesis
ROY L. P. G. JENTJENS,1 LUC J. C. VAN LOON,2 CHRISTOPHER H. MANN,1
ANTON J. M. WAGENMAKERS,2 AND ASKER E. JEUKENDRUP1
1
Human Performance Laboratory, School of Sport and Exercise Sciences, University
of Birmingham, Birmingham B15 2TT, United Kingdom; and 2Department of
Human Biology, Maastricht University, Maastricht 6200 MD, The Netherlands
Received 13 December 2000; accepted in final form 29 March 2001
is often associated
with muscle glycogen depletion (2, 15); therefore, high
preexercise muscle glycogen concentrations are believed to be essential for optimal performance (5, 9, 17).
Because endurance athletes often train twice daily for
several days and may compete on consecutive days,
rapid restoration of muscle glycogen is of crucial importance to optimize recovery.
The complete restoration of muscle glycogen after
prolonged exercise can occur within 24 h, depending on
the degree of glycogen depletion and provided that
sufficient carbohydrates (CHO) are ingested (23, 24). It
has been suggested that muscle glycogen synthesis
after glycogen-depleting exercise occurs in two phases
(31). Initially, there is a period of rapid synthesis of
muscle glycogen that does not require the presence of
insulin and lasts ⬃30–60 min. This early postexercise
recovery period is marked by an exercise-induced permeability of the muscle cell membrane to glucose (18).
GLUT-4 translocation occurs during exercise, and the
increase in the density of GLUT-4 transporters in the
muscle membrane seems to persist for some time after
exercise (27, 36). Together with the continued activation of glycogen synthase (46), this seems to lead to the
initial rapid period of insulin-independent synthesis of
muscle glycogen. The second phase is dependent on
insulin, and glycogen synthesis occurs at a rate that is
10–30% lower than in the first rapid phase (31). When
a CHO supplement is consumed after exercise, blood
glucose and insulin concentrations will rise, and it has
been suggested that this is the mechanism by which
the combined ingestion of CHO and protein can enhance glycogen synthesis (41, 47). Insulin stimulates
both muscle glucose uptake and the activation of glycogen synthase (20), the rate-limiting enzyme in glycogen synthesis. The ability of insulin to stimulate glucose uptake and glycogen synthase activity is even
more pronounced in the first hours after exercise (7,
20). Several studies have attempted to increase insulin
concentrations in the postexercise recovery period to
optimize the rate of muscle glycogen storage (37, 38,
41, 45, 47). Although the pancreatic insulin secretion is
primarily regulated by the blood glucose concentration,
certain amino acids (37, 42) and proteins (41, 47) have
a synergistic effect on the insulin release when administered in combination with a CHO load (38, 39, 41, 47).
Zawadzki et al. (47) observed that the addition of whey
protein to a CHO supplement resulted in an enhanced
rate of glycogen storage during a 4-h recovery period
(47). The authors attributed this effect to a larger
insulin response caused by the ingestion of whey protein. Recently, van Loon et al. (40, 42) investigated
which type, combination, and quantity of free amino
Address for reprint requests and other correspondence: A. E.
Jeukendrup, School of Sport and Exercise Sciences, Univ. of
Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom
(E-mail: [email protected]).
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. Section 1734
solely to indicate this fact.
wheat hydrolysate; carbohydrate-protein drinks; insulin; recovery
FATIGUE DURING PROLONGED EXERCISE
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Jentjens, Roy L. P. G., Luc J. C. van Loon, Christopher H. Mann, Anton J. M. Wagenmakers, and Asker E.
Jeukendrup. Addition of protein and amino acids to carbohydrates does not enhance postexercise muscle glycogen synthesis. J Appl Physiol 91: 839–846, 2001.—Ingestion of a
protein-amino acid mixture (Pro; wheat protein hydrolysate,
leucine, and phenylalanine) in combination with carbohydrate (CHO; 0.8 g 䡠 kg⫺1 䡠 h⫺1) has been shown to increase
muscle glycogen synthesis after exercise compared with the
same amount of CHO without Pro. The aim of this study was
to investigate whether coingestion of Pro also increases muscle glycogen synthesis when 1.2 g CHO 䡠 kg⫺1 䡠 h⫺1 is ingested.
Eight male cyclists performed two experimental trials separated by 1 wk. After glycogen-depleting exercise, subjects
received either CHO (1.2 g 䡠 kg⫺1 䡠 h⫺1) or CHO⫹Pro (1.2 g
CHO 䡠 kg⫺1 䡠 h⫺1 ⫹ 0.4 g Pro 䡠 kg⫺1 䡠 h⫺1) during a 3-h recovery
period. Muscle biopsies were obtained immediately, 1 h, and
3 h after exercise. Blood samples were collected immediately
after the exercise bout and every 30 min thereafter. Plasma
insulin was significantly higher in the CHO⫹Pro trial compared with the CHO trial (P ⬍ 0.05). No difference was found
in plasma glucose or in rate of muscle glycogen synthesis
between the CHO and the CHO⫹Pro trials. Although coingestion of a protein amino acid mixture in combination
with a large CHO intake (1.2 g 䡠 kg⫺1 䡠 h⫺1) increases insulin
levels, this does not result in increased muscle glycogen
synthesis.
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POSTEXERCISE MUSCLE GLYCOGEN RESYNTHESIS
METHODS
Subjects. Eight healthy, trained male cyclists participated
in this study. Subjects trained at least three times per week
for ⬎2 h/day and had been involved in endurance training for
at least 3–5 yr. The protocol and the potential benefits and
risks associated with participation were fully explained to
each subject before they signed an informed consent document. The study was approved by the South Birmingham
Local Research Ethics Committee. Subject characteristics
are listed in Table 1.
Preliminary testing. At least 1 wk before the experimental
trials, the individual maximum power output (Ẇmax) and maximal oxygen consumption (V̇O2 max) were determined by
using an incremental exercise test to exhaustion. Ẇmax
values were used to determine the power output settings
employed in the glycogen depletion protocol. The exercise test
was performed on an electromagnetically braked cycle ergometer (Lode Excalibur Sport, Groningen, The Netherlands), modified to the configuration of a racing bicycle with
adjustable saddle height and handlebar position. Subjects
started with a 3-min warm-up at 95 W, followed by incre-
Table 1. Physical and physiological characteristics
of the subjects
Age, yr
Height, cm
Body mass, kg
V̇O2 max, ml 䡠 kg⫺1 䡠 min⫺1
Ẇmax, W
Ẇmax per kilogram, W/kg
Heart rate maximum, beats/min
27.1 ⫾ 2.6
176.2 ⫾ 1.5
69.6 ⫾ 2.9
63.3 ⫾ 1.5
353.6 ⫾ 13.7
5.1 ⫾ 0.2
191 ⫾ 2
Values are mean ⫾ SE; n ⫽ 8 subjects. V̇O2 max, maximum O2
consumption; Ẇmax, maximum power output.
J Appl Physiol • VOL
mental steps of 35 W every 3 min until exhaustion. Ẇmax
was determined by the following formula [adapted from
Kuipers et al. (25)]
Ẇmax ⫽ Ẇout ⫹ 共t/180兲 ⴱ 35
where Ẇout is the last completed stage and t is the time in the
final stage. Heart rate (HR) was recorded continuously by a
radiotelemetry HR monitor (Polar Vantage NV, Kempele,
Finland). Breath-by-breath measurements were performed
throughout exercise using an Oxycon Alpha automated gas
analysis system (Jaeger, Wuerzberg, Germany). Average inspired and expired volume, O2 consumption (V̇O2), and CO2
production were averaged over eight breaths. V̇O2 was considered maximal (V̇O2 max) when at least two of the following
three criteria were met: 1) a leveling off of V̇O2 with increasing workload (increase of no more than 2 ml 䡠 kg⫺1 䡠 min⫺1), 2)
a HR within 10 beats/min of predicted maximum (HR of 220
beats/min ⫺ age), and 3) a respiratory exchange ratio ⬎ 1.05.
V̇O2 max was calculated as the average V̇O2 over the last 60 s
of the test.
General design. All subjects performed two randomized
“glycogen depletion-restoration” experiments with at least 7
days in between. To minimize differences in resting muscle
glycogen concentration, subjects completed an activity and
diet recall log in which they recorded diet and activity patterns 48 h before the first trial. Subjects were instructed to
follow the same patterns before the second trial. Furthermore, they were asked to avoid vigorous exercise 1 day in
advance of both trials and to fast 10 h before the start of each
experimental exercise trial. To deplete muscle glycogen
stores, subjects exercised on a cycle ergometer with alternating exercise intensities. After cessation of the exercise protocol, a muscle biopsy from the m. vastus lateralis was taken,
and subjects received, in random order, either a CHO plus
wheat hydrolysate amino acid drink (CHO⫹Pro) or a CHOonly drink (CHO). All drinks were lemon flavored to make
the taste comparable in the two trials. One and three hours
after ingestion of the first drink, a second and third biopsy
were taken. Blood samples were collected at registered time
intervals. Throughout the entire 3-h recovery period, subjects
remained seated during which time they could watch television or read.
Experimental protocol. Subjects reported to the Human
Performance Laboratory at the University of Birmingham
after an overnight fast (ⱖ10 h). Muscle glycogen depletion
was induced as follows: subjects performed a graded exercise
test to exhaustion as described before (Ẇmax protocol); they
then rested for 10 min and subsequently performed an intermittent exercise protocol as described by Kuipers et al. (25).
In short, after a 10-min warm-up period at 50% Ẇmax,
subjects were instructed to cycle 2-min block periods at
alternating workloads of 90 and 50% Ẇmax. When the subject was unable to complete a 2-min block at 90% Ẇmax (i.e.,
subjects were unable to maintain a cadence of 60 rpm),
despite encouragement from the experiment leader, the
workload was lowered to 80% Ẇmax. Again subjects continued cycling until they were unable to complete a 2-min block
at 80% Ẇmax, after which the high-intensity block was
reduced to 70% Ẇmax. The exercise was stopped when the
2-min block at 70% Ẇmax could not be completed. This
protocol has been shown to result in very low muscle glycogen
concentrations (26). All tests were performed under normal
and standard environmental conditions (22–25°C dry bulb
temperature and 50–60% relative humidity). During the
exercise tests, subjects were cooled with standing floor fans to
minimize thermal stress, and water was available ad libitum.
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acids or protein sources would maximize the insulin
response when coingested with CHO. It was shown
that a mixture of wheat protein hydrolysate, free
leucine, and free phenylalanine, when added to a CHO
drink (0.8 g CHO 䡠 kg⫺1 䡠 h⫺1), resulted in higher insulin
concentrations and an increased glycogen synthesis
rates compared with a CHO-only drink. In this study
(41), drinks were ingested at regular intervals (30
min), in contrast to the study of Zawadzki et al. (47), in
which drinks were given as two large boluses (120-min
intervals).
van Loon et al. (41) showed that, when the rate of
CHO intake was increased to 1.2 g 䡠 kg⫺1 䡠 h⫺1, this also
resulted in significantly higher muscle glycogen synthesis rates compared with the ingestion of 0.8
g 䡠 kg⫺1 䡠 h⫺1. These findings suggest that maximal glycogen synthesis rates were not reached when 0.8 g
CHO 䡠 kg⫺1 䡠 h⫺1 was ingested.
It is not known whether the addition of a proteinamino acid mixture to a larger amount of CHO would
further increase glycogen synthesis after exercise.
Therefore, the purpose of the present study was to
investigate whether coingestion of an insulinotropic
amino acid mixture and a high rate of CHO intake (1.2
g 䡠 kg⫺1 䡠 h⫺1) provided at 30-min intervals would increase the rate of muscle glycogen resynthesis in the
postexercise recovery period compared with a highCHO intake only.
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POSTEXERCISE MUSCLE GLYCOGEN RESYNTHESIS
Fig. 1. Schematic representation for each testing day.
J Appl Physiol • VOL
Table 2. Composition of beverages
Trial
CHO
Leucine, g/l
Phenylalanine, g/l
Wheat hydrolysate, g/l
Glucose (dextrose monohydrate), g/l
Maltodextrin, g/l
Sodiumsaccharinate (25% wt/wt), g/l
Citric acid (50% wt/wt), g/l
Lemon flavor, ␮l
Water, liters
94.36*
85.71
0.8
3.6
400
up to 1
CHO ⫹ Pro
14.28
14.28
28.57
94.36*
85.71
0.8
3.6
400
up to 1
CHO, carbohydrate only beverage; CHO ⫹ Pro, carbohydrate plus
protein mixture beverage. * After correction for the extra H2O molecule in dextrose monohydrate.
polypeptide derived from wheat protein with a medium chain
length of 11 amino acids. The exact amino acid profile of the
wheat hydrolysate has been described previously (42). The
maltodextrins used had a medium chain length of 10–16
glycosyl units. To make the taste comparable in both trials,
0.8 g sodium-saccharinate solution (25% wt/wt), 3.6 g citric
acid solution (50% wt/wt; Quest), and 400 ␮l of lemon flavoring (Supercook, Leeds, UK) were added for each liter of drink.
Analyses. Blood samples were collected into prechilled
tubes containing 200 ␮l of 0.2 M EDTA and centrifuged at
1,500 g for 5 min at 4°C. Aliquots of plasma were stored at
⫺70°C until further analysis for glucose and insulin. Glucose
(Glucose K kit, 07260102: Sigma-Aldrich, Dorset, UK) was
analyzed on a COBAS BIO semiautomatic analyzer (La
Roche, Basel, Switzerland). Plasma insulin was determined
by radioimmunoassay by using a commercially available kit
(insulin 125I-RIA 100 kit, ICN Pharmaceuticals, Costa Mesa,
CA). The intra-assay coefficient of variation for glucose and
insulin was ⬍2%.
Muscle biopsy samples (⬃50 mg wet wt) were freeze dried
for 2 days at approximately ⫺40°C and for 0.5 days at room
temperature. Then the muscle samples were dissected free of
connective tissue, visible fat, and blood using a light microscope. Each muscle sample was minced with a scalpel, and
⬃3 mg were weighed (Mettler, PM 6400, accuracy of 0.001
mg) for determination of muscle glycogen concentration.
Muscle samples were hydrolyzed in 1 M HCl at 100°C for 3 h.
After cooling down to room temperature, 175 ␮l Tris 䡠 KOH
neutralization mixture (1.44 g Tris and 12 g KOH per 100 ml
distilled water) was added. Samples were centrifuged at
9,000 rpm for 10 min at 4°C, and muscle glycogen concentrations were determined as glucose residues on the Cobas Fara
semiautomatic analyser (LaRoche).
Calculations. Muscle glycogen synthesis rate was calculated from the following equation: muscle glycogen synthesis
rate ⫽ (GtA ⫺ GtB)/⌬t, where GtA and GtB are the muscle
glycogen concentrations at A and B hours postexercise (i.e.,
Gt1 ⫺ Gt0, Gt3 ⫺ Gt1, and Gt3 ⫺ Gt0, respectively), and ⌬t is
the time between the two biopsies. Muscle glycogen synthesis
rates were expressed as millimoles of glycosyl units per
kilogram of dry weight (dw) per hour (mmol 䡠 kg dw⫺1 䡠 h⫺1).
Questionnaires. Subjects were asked to fill out a questionnaire immediately after the exercise test (before the first
beverage was received) and every hour thereafter. This questionnaire contained questions regarding the presence of gastrointestinal (GI) problems at that moment and addressed
the following complaints: stomach problems, GI cramping,
bloated feeling, diarrhea, nausea, dizziness, headache, belching, urge to urinate and/or defecate. The items were scored on
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After cessation of the depletion exercise protocol, subjects
were allowed to take a shower, and, within 20 min postexercise, a 50- to 100-mg muscle sample was then obtained from
the vastus lateralis using the percutaneous needle biopsy
technique modified to include suction (1). After the skin was
cleaned and 5-ml local anesthesia (2% lignocaine) was administered, a 4- to 6-mm incision in the skin and muscle
fascia was made 12–20 cm above the patella on the lateral
side of the vastus lateralis. The obtained muscle samples
were immediately frozen in liquid nitrogen and stored at
⫺70°C for later analysis of muscle glycogen content.
A 21-gauge Teflon catheter (Baxter, Norfolk, UK) was then
inserted in an antecubital vein for blood sampling. A resting
blood sample (10 ml) was collected, stored on ice, and later
centrifuged. The catheter was kept patent by flushing with
0.5 ml of isotonic saline (0.9%; Baxter). Immediately after the
resting blood sample was taken (t ⫽ 0; Fig. 1), subjects
received the first bolus of test drink. Blood samples were
taken at 30-min intervals for determination of plasma glucose and insulin until t ⫽ 180 min. A second and third muscle
biopsy sample were taken at t ⫽ 60 min using the same
incision and at t ⫽ 180 min from the contralateral leg. The
second biopsy was obtained by angling the biopsy needle at
least 3 cm distal to the initial incision. To reduce local tissue
inflammation and/or membrane disruption after repeated
muscle biopsy sampling from one leg, the third muscle sample was taken from the contralateral leg (10).
Beverages. At t ⫽ 0, 30, 60, 90, 120, and 150 min, subjects
received a beverage volume of 3.5 ml/kg to ensure a given
dose of 1.2 g 䡠 kg⫺1 䡠 h⫺1 of CHO (50% glucose and 50% maltodextrin) in the CHO trial and 1.2 g 䡠 kg⫺1 䡠 h⫺1 of CHO (50%
glucose and 50% maltodextrin) and 0.4 g 䡠 kg⫺1 䡠 h⫺1 of a
protein hydrolysate and amino acid mixture in the CHO⫹Pro
trial. The protein hydrolysate and amino acid mixture consisted of a wheat protein hydrolysate (50% mass) and two
free amino acids: leucine (25% mass) and phenylalanine (25%
mass). This protein hydrolysate and amino acid mixture has
previously been reported to result in high-insulin responses
(40, 42) and increased glycogen synthesis rates when coingested with moderate amounts of CHO (41). The compositions of the test drinks are presented in Table 2.
Glucose (dextrose monohydrate) was obtained from Cerestar (Manchester, UK). A correction factor was applied to
correct for the difference in molecular weight between glucose and dextrose monohydrate by multiplying the amount of
glucose by 1.1 (⫽ 198/180). Maltodextrin was obtained from
Nutriforce (Sittard, The Netherlands), amino acids were obtained from Sigma Aldrich (Gillingham, UK), and protein
hydrolysate (Hyprol DEV 4107) was prepared by Quest
(Naarden, The Netherlands). The protein hydrolysate is a
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POSTEXERCISE MUSCLE GLYCOGEN RESYNTHESIS
a 10-point scale (1 ⫽ not at all, 10 ⫽ very, very much). The
severity of the GI problems was divided into two categories:
a score ⬍6 (i.e., 1–5) or a score ⱖ6 (i.e., 6–10). Because there
were no GI problems immediately after exercise, only the
results of the last three questionnaires are reported. For each
complaint, a maximum of 24 (8 subjects ⫻ 3 h) could be
scored.
Statistics. All data are expressed as means ⫾ SE. Two-way
(time ⫻ treatment) ANOVA for repeated measurements was
used to compare differences in glycogen, plasma insulin, and
glucose concentration. When these analyses revealed significant differences, a Tukey’s post hoc test was used to locate
the difference. Plasma glucose and insulin responses were
calculated as areas under the curve. Statistical analysis of
these data were calculated by using a paired Student’s t-test.
Differences were considered to be significant at P ⬍ 0.05.
RESULTS
J Appl Physiol • VOL
Fig. 2. Glucose (A) and insulin (B) concentrations during 3-h recovery period. CHO, carbohydrate-only trial; CHO⫹Pro, carbohydrate
plus protein mixture trial. Values are means ⫾ SE; n ⫽ 8 subjects.
* P ⬍ 0.05 for CHO trial compared with CHO⫹Pro trial.
the curve for glucose calculated for each time interval
(0–60, 60–180, and 0–180 min) did not differ between
the two trials (Fig. 3A).
Muscle glycogen. Muscle glycogen concentrations
were obtained from only seven subjects because of
technical problems. The muscle glycogen concentration
immediately after exercise was similar between the
two trials [106 ⫾ 19 (CHO) vs. 176 ⫾ 31 mmol/kg dw
(CHO⫹Pro)]. Over the entire 3-h recovery period, muscle glycogen concentration increased to 225 ⫾ 22
mmol/kg dw in the CHO trial and to 252 ⫾ 48 mmol/kg
dw in the CHO⫹Pro trial (Table 3). There were no
significant differences in muscle glycogen synthesis
rates between the CHO and CHO⫹Pro trials for either
the first 60 min or between 60 and 180 min postexercise. No differences in glycogen synthesis rates were
found between the first 60 min and the final 120 min of
recovery [35 ⫾ 22 (0–60 min) vs. 31 ⫾ 10 mmol 䡠 kg
dw⫺1 䡠 h⫺1 (60–180 min)].
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Exercise trials. After subjects had performed a
graded exercise to exhaustion, they cycled on average
the following number of 2-min blocks: 11.3 ⫾ 1.7, 7.8 ⫾
1.3, 8.4 ⫾ 1.5, and 27.8 ⫾ 3.4 at 90, 80, 70, and 50%
Ẇmax, respectively. The average work rate corresponding to 90, 80, 70, and 50% Ẇmax was 318 ⫾ 12, 283 ⫾
11, 248 ⫾ 10, and 177 ⫾ 7 W, respectively. The total
exercise duration and the average workload in the
CHO and CHO⫹Pro trials were not significantly different (143 ⫾ 10 vs. 128 ⫾ 11 min and 229 ⫾ 10 vs.
232 ⫾ 10 W, respectively). Over the entire exercise
period, the average HR was 158 ⫾ 1.5 beats/min, which
corresponded to 83 ⫾ 1% maximum heart rate.
Insulin and glucose. The changes in plasma glucose
and insulin concentrations during the recovery period
are shown in Fig. 2, A and B, respectively. Plasma
insulin concentration immediately after exercise was
9.1 ⫾ 1.8 ␮U/ml in the CHO trial and 9.7 ⫾ 0.9 ␮U/ml
in the CHO⫹Pro trial. In both trials, plasma insulin
levels increased during the first 120 min and remained
relatively constant during the final 60 min of recovery.
During the final 60 min of recovery, plasma insulin
concentration was significantly higher in the
CHO⫹Pro trial compared with the CHO trial.
The addition of the protein hydrolysate and amino
acid mixture resulted in a significantly higher insulin
response (expressed as area under the curve) between
60–180 min and 0–180 min of recovery compared with
the CHO trial (Fig. 3B) (12.9 ⫾ 2.5 vs. 6.1 ⫾ 0.8 mU/ml
at 120 min and 14.7 ⫾ 2.9 vs. 7.3 ⫾ 0.9 mU/ml at 180
min, respectively; P ⬍ 0.05). However, during the first
60 min postexercise, the area under the curve for
insulin was not significantly different between the two
trials [1.1 ⫾ 0.2 (CHO) vs. 1.8 ⫾ 0.4 mU/ml (CHO⫹Pro)
at 60 min].
There was no significant difference between the two
trials in plasma glucose concentration at any time
point during the recovery period. Plasma glucose concentration was low immediately after exercise (3.3–3.5
mmol/l) but increased rapidly during the first hour
(6.5–7.2 mmol/l), after which glucose levels remained
stable in the second hour and finally declined to values
of 5.1 and 5.6 mmol/l at 180 min postexercise for the
CHO and CHO⫹Pro trial, respectively. The area under
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Table 4. Gastrointestinal problems during 3 h
of recovery after glycogen-depleting exercise
CHO ⫹ Pro
CHO
Complaint
0–5
6–10
0–5
6–10
Stomach problems
Nausea
Dizziness
Headache
Flatulence
Urge to urinate
Urge to defecate
Belching
Stomach burn
Bloated feeling
Stomach cramps
Intestinal cramps
Diarrhea
24
20
24
23
24
22
24
21
24
22
24
24
24
0
4
0
1
0
2
0
3
0
2
0
0
0
24
24
24
24
24
20
24
24
24
24
24
24
24
0
0
0
0
0
4
0
0
0
0
0
0
0
0–5 and 6–10 are scores of severity of complaint where 1 ⫽ not at
all and 10 ⫽ very, very much. For each complaint, a maximum of 24
could be scored (8 subjects ⫻ 3 h).
GI complaints. In general, subjects reported nonsevere GI symptoms (a score ⬍ 5). However, two subjects
reported a high score regarding nausea, belching, and
bloated feeling after consuming the CHO⫹Pro beverages (Table 4).
DISCUSSION
Previous studies have shown that the addition of
certain amino acids and/or proteins to a CHO supplement can increase glycogen synthesis rates as a result
of an enhanced insulin response (41, 47). The major
finding of the present study was that the ingestion of
an insulinotropic protein hydrolysate and amino acid
Table 3. Mean muscle glycogen contents and synthesis rates postexercise
Synthesis Rate, mmol 䡠 kg dw⫺1 䡠 h⫺1
Glycogen Content, mmol/kg dw
CHO
CHO ⫹ Pro
0h
1h
3h
0–1 h
1–3 h
0–3 h
106 ⫾ 19
176 ⫾ 31
141 ⫾ 25
211 ⫾ 41
225 ⫾ 22
252 ⫾ 48
35 ⫾ 34
35 ⫾ 31
42 ⫾ 9
21 ⫾ 16
40 ⫾ 10
25 ⫾ 16
Values are mean ⫾ SE; n ⫽ 7 subjects. dw, Dry weight.
J Appl Physiol • VOL
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Fig. 3. Glucose (A) and insulin (B) responses during 3-h recovery
period. Values are means ⫾ SE; n ⫽ 8 subjects. * P ⬍ 0.05 for CHO
trial compared with CHO⫹Pro trial.
mixture, in combination with a large CHO intake (1.2
g 䡠 kg⫺1 䡠 h⫺1), provided at 30-min intervals, did not affect the rate of muscle glycogen synthesis during the
early postexercise recovery period. Although the
CHO⫹Pro supplement resulted in significantly higher
insulin concentrations, a difference in the glucose response or in the rate of muscle glycogen storage between the two trials was not observed. Furthermore,
the higher prevalence of GI discomfort after ingestion
of CHO⫹Pro drinks may limit their usefulness as recovery drinks, at least in some subjects.
Zawadzki et al. (47) found that the insulin rise and
glycogen repletion rate was 39% greater with a combined CHO-protein supplement compared with a CHO
supplement only. This study has been criticized by
several investigators because it did not include a control trial (isoenergetic amount of CHO intake) (6, 34,
35). Previous studies (6, 34, 35) found similar muscle
glycogen resynthesis rates for both CHO and isoenergetic CHO⫹Pro (⫹fat) supplements. They suggested
that the total amount of energy consumed in the postexercise period is an important factor for muscle glycogen synthesis. The present study did not examine the
effects of isoenergetic CHO and CHO⫹Pro feedings.
However, it is also possible that the increased glycogen
synthesis rate was indeed the result of elevated insulin
levels caused by the addition of protein rather than a
difference in energy intake between the two trials (41,
42, 47). Recently, van Loon et al. (41) showed that the
addition of an insulinotropic protein hydrolysate and
amino acid mixture to a CHO beverage increased mus-
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POSTEXERCISE MUSCLE GLYCOGEN RESYNTHESIS
J Appl Physiol • VOL
did not measure gastric emptying, and, therefore, we
could not investigate the effects of gastric emptying on
muscle glycogen synthesis. Adding a smaller amount of
protein might have reduced a potential negative effect
on the rate of gastric emptying and could have increased the rate of appearance of glucose into the
circulation.
There is also convincing evidence that this limitation
is not located at the muscular level (14). Hansen et al.
(14) reported an initial glycogen synthesis rate of 185
mmol 䡠 kg dw⫺1 䡠 h⫺1 after a prolonged infusion of supraphysiological concentrations of glucose and insulin
after exercise. Although this study was based on only
two subjects, the rate of muscle glycogen storage was
far above the maximal glycogen synthesis rate of 40–50
mmol 䡠 kg dw⫺1 䡠 h⫺1 often found in studies where glucose was ingested orally after glycogen-depleting exercise (4, 8, 11, 30, 41). Therefore, more likely the rate of
muscle glycogen synthesis is limited by the rate of
digestion and absorption of CHO by the intestine and
subsequent transport of glucose into the blood stream
regulated by the liver. It has been suggested that the
upper limit for glucose absorption in human is ⬃1
g/min (22, 32), which may be slightly higher in the
postexercise state (13). Assuming an active muscle
mass of 10 kg during cycling (12), the maximal rate of
muscle glycogen storage after oral glucose consumption should be in the range of 0.3–0.4 g/min. Thus the
maximal glycogen synthesis rate in the leg seems to be
60–65% lower than the maximal absorption rate by the
gut, which indicates that part of the absorbed glucose
must be extracted by other tissues (i.e., other muscle
groups, fat tissue, or the liver). Therefore, the transport capacity of the intestine for glucose cannot be the
only factor that determines the (maximal) rate of muscle glycogen synthesis.
Although the amount of CHO intake seems to be a
very important factor determining the rate of glycogen
synthesis, there is still no conclusive answer as to how
much CHO needs to be consumed in the postexercise
recovery phase to maximize the rate of glycogen synthesis. Initially, Blom et al. (3) demonstrated that
increasing CHO intake from 0.35 to 0.7 g 䡠 kg⫺1 䡠 h⫺1 did
not result in an increased muscle glycogen storage
rate. Ivy et al. (21) found similar glycogen storage rates
during the first 4 h of recovery when a CHO supplement of either 0.75 or 1.5 g 䡠 kg⫺1 䡠 h⫺1 was provided
(19.6 vs. 22.0 mmol䡠kg dw⫺1 䡠h⫺1). However, in both studies, CHO drinks were supplemented at 2-h intervals. It
was suggested that this feeding protocol may not have
adequately increased and maintained blood glucose
and insulin levels for 2 h (16), which could explain the
discrepancy between these results and those of others
(8, 11, 30, 41). Several studies have reported higher
glycogen synthesis rates (between 40 and 45 mmol 䡠 kg
dw⫺1 䡠 h⫺1) when 1.0–1.85 g 䡠 kg⫺1 䡠 h⫺1 of CHO were
consumed more frequently (15- to 60-min intervals)
during a 3- to 5-h recovery period (8, 11, 30, 41). The
glycogen synthesis rates found in the present study are
consistent with previously reported results of several
other studies in which almost similar amounts of CHO
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cle glycogen synthesis rates by more than twofold. The
increased rate of muscle glycogen synthesis after exercise was attributed to an increased clearance of glucose
by the muscle as a result of higher insulin levels (41,
46). In the present study, we have used the same
protein hydrolysate and amino acid supplement as was
used by van Loon et al. (40, 41) but a higher amount of
CHO. Ingestion of 1.2 g CHO 䡠 kg⫺1 䡠 h⫺1 in this study
resulted in a similar insulin response as in the study by
van Loon et al. (40). Addition of the protein hydrolysate
and amino acids resulted in an even larger insulin
response (Fig. 3B). However, despite this increase in
plasma insulin levels, we did not find an effect on
glycogen synthesis, which seems to be in contrast to
earlier findings by Zawadzki et al. (47). There may be
two reasons for this apparent discrepancy. In this
study, a larger amount of CHO (1.2 g CHO 䡠 kg⫺1 䡠 h⫺1)
was ingested compared with that in the study of
Zawadzki et al. (47) (0.77 g 䡠 kg⫺1 䡠 h⫺1), and feeding was
more frequent (every 30 min compared with 120 min).
The present data, together with those of van Loon et
al. (41), suggest that insulin is not the rate-limiting
factor for muscle glycogen synthesis when total CHO
intake is high and provided at 30-min intervals. Recent
work from van Hall et al. (38) has shown that, despite
higher insulin levels with CHO-protein ingestion compared with CHO ingestion alone, leg glucose uptake
was not increased in the CHO-protein trial. Similar to
the results of the present study, van Hall et al. (38)
found no beneficial effect of the increased insulin levels
on muscle glycogen synthesis when CHO was ingested
at rates of 1.0 g 䡠 kg⫺1 䡠 h⫺1. The authors suggested that
a relatively low insulin level may already elicit maximal achievable GLUT-4 migration to the cell membrane and glycogen synthesis rates when CHO intake
is ample. Together, the present study and other recent
studies (38, 41) suggest that the availability of substrate (i.e., glucose) is the main limiting factor for
glycogen synthesis.
The availability of glucose is dependent on the rate of
gastric emptying and intestinal absorption of the ingested glucose, glucose output by the liver, and glucose
entry into the muscle. Studies that have examined
gastric emptying in relation to exogenous CHO oxidation have shown that the rate of gastric emptying is not
the limiting step in the oxidation of the oral ingested
glucose (29, 33). It is, therefore, unlikely that the rate
of gastric emptying will limit the rate of glycogen
synthesis when only CHO is ingested. However, it is
well known that the rate of gastric emptying is affected
by the energy density of the food consumed (43, 44).
The higher energy density in the CHO⫹Pro trial may
have confounded the results in the present study by
inhibiting gastric emptying. This may have slowed
down the rapid delivery of glucose to the muscle and
amino acids to simulate the pancreas for insulin secretion. If gastric emptying would hinder the delivery of
glucose to the muscle, it is possible that less glycogen
synthesis would have occurred in the first phase of
glycogen synthesis when contraction-induced GLUT-4
migration was still present. In this study, however, we
POSTEXERCISE MUSCLE GLYCOGEN RESYNTHESIS
were ingested (6, 19, 28, 35, 38, 41). The glycogen
concentrations found immediately after exercise were
in good agreement with the results reported by van
Loon et al. (41), where subjects performed a similar
amount of work (exercise duration ⬎90 min at workloads varying between 50 and 90% Ẇmax).
Summary. The results of the present study do suggest that a further increase in the insulin concentration by additional supplementation of protein and
amino acids does not increase the rate of glycogen
synthesis when CHO intake is sufficient and supplemented at regular intervals of ⱕ30 min. Insulin can,
therefore, be excluded as the limiting factor for glycogen synthesis. The total amount of glucose intake postexercise, on the other hand, seems to play a more
important role when maximal rates of muscle glycogen
synthesis are required. An intake of 1.2 g 䡠 kg⫺1 䡠 h⫺1 or
more seems to be required to achieve the maximal
glycogen resynthesis rate.
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