Postexercise nutrient intake timing in humans is critical to
recovery of leg glucose and protein homeostasis
Deanna K.
Levenhagen1,
Jennifer D.
Gresham1,
Michael G.
Carlson3,
David
J.
Maron3,
Myfanwy J.
Borel1, and
Paul J.
Flakoll1,2
Departments of 1 Surgery, 2 Biochemistry, and
3 Medicine, Vanderbilt University Medical Center, Nashville,
Tennessee 37232
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ABSTRACT |
Although the importance
of postexercise nutrient ingestion timing has been investigated for
glycogen metabolism, little is known about similar effects for protein
dynamics. Each subject (n = 10) was studied twice, with the
same oral supplement (10 g protein, 8 g carbohydrate, 3 g
fat) being administered either immediately (EARLY) or 3 h (LATE)
after 60 min of moderate-intensity exercise. Leg blood flow and
circulating concentrations of glucose, amino acids, and insulin were
similar for EARLY and LATE. Leg glucose uptake and whole body glucose
utilization (D-[6,6-2H2]glucose)
were stimulated threefold and 44%, respectively, for EARLY vs. LATE.
Although essential and nonessential amino acids were taken up by the
leg in EARLY, they were released in LATE. Although proteolysis was
unaffected, leg
(L-[ring-2H5]phenylalanine)
and whole body (L-[1-13C]leucine) protein
synthesis were elevated threefold and 12%, respectively, for EARLY vs.
LATE, resulting in a net gain of leg and whole body protein. Therefore,
similar to carbohydrate homeostasis, EARLY postexercise ingestion of a
nutrient supplement enhances accretion of whole body and leg protein,
suggesting a common mechanism of exercise-induced insulin action.
synthesis; deposition; amino acids; exercise
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INTRODUCTION |
SYNTHESIS OF GLYCOGEN AND
PROTEIN is essential for skeletal muscle recovery from the
catabolic events of exercise. Glycogen is broken down and used during
exercise as energy for muscle contraction. Because of the damage of
muscle proteins and the diversion of amino acids and energy away from
the events of protein synthesis during exercise, there also is a need
for increased postexercise protein repair and synthesis. It has been
well established that the timing of carbohydrate intake after exercise
significantly influences postexercise carbohydrate homeostasis and
recovery (22, 43). For example, when carbohydrate
supplements were provided to twelve male cyclists several minutes after
exercise, muscle glycogen storage was more rapid than when the same
supplement was consumed 2 h after exercise (22).
Although the importance of timing for carbohydrate intake after
exercise has been demonstrated, little information exists as to the
influence of timing for postexercise protein intake.
Exercise induces a greater sensitivity and responsiveness to the events
controlled by insulin (44). In vitro and in vivo studies
have demonstrated increased insulin-mediated glucose uptake in response
to muscle contraction (6, 9, 10, 28, 34, 35, 48).
Furthermore, exercise has been shown to benefit patients with
non-insulin-dependent diabetes mellitus by lowering postexercise blood glucose, lowering basal and postprandial insulin concentrations, improving insulin sensitivity, and reducing glycosylated hemoglobin levels (10). These observations potentially could be
explained by increased blood flow (9) enhancing nutrient
availability to tissues for the same magnitude of insulin. However,
even at constant receptor and nutrient concentrations,
insulin-stimulated glucose and amino acid transport have been shown to
be increased by muscular contraction (48).
A vital role also has been demonstrated for insulin in the regulation
of protein dynamics (12). Therefore, if exercise
stimulates insulin sensitivity and responsiveness, the timing of
postexercise nutrient supplementation may alter protein dynamics.
However, the influence of postexercise nutrient supplementation on
protein dynamics in humans is undefined. Therefore, the objective of
this investigation was to examine how the timing of postexercise
nutrient ingestion affects whole body and leg protein dynamics in
healthy adults.
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SUBJECTS AND METHODS |
Subject selection.
Ten healthy adult subjects (5 males and 5 females), aged 20-41 yr
and within 25% of ideal body weight based on Metropolitan Life
Insurance Company tables (27), were selected (see Table 1)
and screened for participation in a metabolic study at the Vanderbilt
University Medical Center, Nashville, TN. Each subject was provided
with an explanation of the study, and informed written consent was
obtained for procedures to be performed at the General Clinical
Research Center (GCRC). The experimental protocols and procedures were
approved by the Institutional Review Board of Vanderbilt University
Medical Center.
Each subject underwent a complete physical examination and provided a
full medical history. None of the subjects had any apparent hepatic,
pulmonary, thyroid, renal, or metabolic dysfunction. Female subjects
were not pregnant, as determined by a pregnancy test, and were
premenopausal with regular menstrual cycles. Females were studied
between 3 and 10 days after the onset of menses (follicular phase) to
reduce experimental variability. Each subject's body density was
determined by hydrostatic weighing, and body fat and lean masses were
calculated using equations for either Caucasians or African Americans,
as previously described (24). A maximal exercise test was
conducted in which each subject used a recumbent stationary cycle
(Ergometrics 800, Ergoline, Bandhagen, Sweden) to perform incremental
(+25 W/min) exercise until exhaustion. A respiratory exchange ratio of
>1.0 and an increase in O2 uptake (
O2) of <0.2 l/min over the previous
work rate were the criteria used for maximal
O2
(
O2 max). Energy expenditure (EE) was
determined during rest and during exercise using indirect calorimetry
(Sensormedics 2900 Metabolic Cart, Yorba Linda, CA).
Metabolic study protocol.
For 3 days before the metabolic studies, subjects received their meals
from the dietary kitchen of the GCRC to maintain consistency between
preexercise body nutrient stores. Energy intake was kept at maintenance
levels on the basis of the Harris Benedict equation and each subject's
gender, height, weight, and activity level.
On metabolic study days, subjects were admitted to the GCRC after an
overnight fast (>12 h). To obtain samples of venous blood draining
from the leg, a 5-French sheath was introduced into the femoral vein
under local anesthesia (1% xylocaine infiltration), and the distal tip
of the sheath was positioned, using fluoroscopy in the external iliac
vein, a few centimeters above the inguinal ligament. Indwelling
catheters also were placed in a heated superficial hand vein for
arterialized blood sampling and in the antecubital vein of the
nondominant arm for infusion of stable isotopic tracers. The
catheterized hand was placed in a heated thermoplastic box, with the
temperature adjusted to 55°C for complete arterialization of blood
samples (1).
After a collection of blood and breath samples to determine
isotopic backgrounds (
210 min), a bolus infusion of
[13C]NaHCO3 (0.24 mg/kg),
D-[6,6-2H2]glucose (3.6 mg/kg),
L-[1-13C]leucine (14.4 µmol/kg), and
L-[ring-2H5]phenylalanine
(3.6 µmol/kg) was given to prime the carbon dioxide, glucose,
leucine, and phenylalanine pools, respectively. Subsequently, a
continuous infusion of
D-[6,6-2H2]glucose (0.06 mg · kg
1 · min
1),
L-[1-13C]leucine (0.24 µmol · kg
1 · min
1),
L-[ring-2H5]phenylalanine
(0.06 µmol · kg
1 · min
1),
and [2H5]glycerol (0.12 µmol · kg
1 · min
1) was
initiated and continued throughout the study. Each metabolic study
consisted of four periods (Fig. 1):
1) a 120-min equilibration period; 2) a 30-min
basal sampling period; 3) a 60-min exercise period; and
4) either a 180-min or 360-min recovery period. During the
60-min exercise period, subjects exercised on a recumbent bicycle at
60% of
O2 max as determined by
indirect calorimetry and heart rate measures.

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Fig. 1.
The experimental design consisted of equilibration,
basal, exercise, and postexercise recovery periods. During the recovery
period of either 180 or 360 min, isotopic tracers of phenylalanine,
leucine, glucose, and glycerol were infused to measure leg and whole
body kinetics. Each subject was studied twice, and an oral nutrient
supplement (10 g protein, 8 g carbohydrate, and 3 g fat) was
administered either immediately after exercise (EARLY) or 3 h
after exercise (LATE). Blood and breath samples ( ) were
taken during the basal period and for 3 h after nutrient intake.
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Arterial and venous blood samples were taken every 15 min during the
basal period and every 30 min for 180 min after postexercise nutrient
intake for determination of hormone and metabolite concentrations, as
well as isotopic enrichments. Simultaneously, breath samples were
collected from each subject in a Douglas bag, and duplicate 20-ml
samples were placed into nonsiliconized evacuated glass tubes for the
determination of breath 13CO2 enrichment. Leg
blood flow measurements were determined by plethysmography (model 2560 with URI/CP software version 3.0; UFI, Morro Bay, CA)
(31). Finally, carbon dioxide production,
O2, and EE were determined
throughout each period by indirect calorimetry (Sensormedics 2900 Metabolic Cart, Palo Alto, CA).
Experimental design.
Each subject was studied twice. A nutrient supplement containing
10 g protein, 8 g carbohydrate, and 3 g lipid (Jogmate;
Pharmavite, Mission Hills, CA) was administered either immediately
(EARLY) or 3 h after the conclusion of exercise (LATE). The
majority of protein, carbohydrate, and fat in the nutrient supplement
was derived from casein, regular sugar, and milk fat, respectively. The
order of treatment administration was random. A 4-wk "washout" period was maintained between metabolic studies to allow isotopic tracer clearance and to ensure that the female participants were in the
follicular phase of their menstrual cycle. Subjects were instructed to
maintain daily exercise activity, dietary intake, and a constant body
weight for 2 wk before each test day so that they remained similar
between treatments.
Analytical procedures.
Blood samples were collected into Venoject tubes containing 15 mg
Na2EDTA (Terumo Medical, Elkton, MD). A 1-ml aliquot of whole blood from each sampling site was deproteinized with 3 ml of 4%
perchloric acid for determination with enzymatic methods of whole blood
lactate, glycerol, and glutamine concentrations (25, 26).
In addition, 3 ml of blood were transferred to a tube containing EDTA
and reduced glutathione, with the plasma stored at
80°C for later
measurement of plasma epinephrine and norepinephrine concentrations by
HPLC (17). The remaining blood was spun in a refrigerated
(4°C) desktop centrifuge (Beckman Instruments, Fullerton, CA) at
3,000 rpm for 10 min to obtain the plasma, which was stored at
80°C
for later analysis. Plasma glucose concentrations were determined by
the glucose oxidase method (Model II Glucose Analyzer; Beckman
Instruments, Fullerton, CA).
Immunoreactive insulin was determined in plasma with a double-antibody
system (29). Plasma aliquots for glucagon determination were placed in tubes containing 25 kallikrein-inhibitor units of
aprotinin (Trasylol; FBA Pharmaceutical, New York, NY) and were later
measured by established radioimmunoassay with a double-antibody system
modified from the method of Morgan and Lazarow (29) for insulin. Insulin and glucagon antisera and standards, as well as
125I-labeled hormones, were obtained from RL Gingerich
(Linco Research, St. Louis, MO). A Clinical Assays Gammacoat
Radioimmunoassay kit (Travenol-GenTech, Cambridge, MA) was used to
measure plasma cortisol concentrations. Plasma amino acid
concentrations were determined by reversed-phase HPLC after
derivatization with phenylisothiocyanate (20). Individual
amino acids were also placed into groups for analysis purposes. These
groups included branched-chain amino acids (BCAA), the sum of leucine,
isoleucine, and valine; essential amino acids (EAA), the sum of
arginine, histidine, isoleucine, leucine, lysine, methionine,
phenylalanine, threonine, tryptophan, and valine; total amino acids
(TAA), the sum of all individual amino acids; and nonessential amino
acids (NEAA), the difference between TAA and EAA.
After deproteinization with Ba(OH)2 and ZnSO4
and elution over cation and anion resins, plasma
D-[6,6-2H2]glucose enrichment was
determined by gas chromatography-mass spectrometry (GC-MS) according to
the method of Bier et al. (5). Plasma enrichments of
[13C]leucine, [13C]ketoisocaproate (KIC),
and [ring-2H5]phenylalanine were
determined using GC-MS. Plasma was deproteinized with 4% perchloric
acid, and the supernatant was passed over a cation exchange resin to
separate the keto and amino acids. The keto acids were further
extracted with methylene chloride and 0.5 M ammonium hydroxide
(30). After drying under nitrogen gas, both the keto and
amino acid fractions were derivatized (37) with
N-methyl-N-(t-butyldimethylsilyl)-trifluoroacetamide
containing 1% t-butyldimethylchlorosilane (MtBSTFA + 1% t-BDMCS; Regis Technologies, Morton Grove, IL). The
derivatized samples were then analyzed with GC-MS (Hewlett-Packard
5890a GC and 5970 MS, San Fernando, CA) for plasma leucine and KIC
enrichments. For determination of
[2H5]glycerol enrichment, plasma was
deproteinized with 4% perchloric acid, and the supernatant was passed
over cation and anion exchange resins. The eluate was dried overnight
at 50°C, the glycerol fraction was derivatized with MtBSTFA + 1% t-BDMCS, and the derivatized samples were analyzed by
GC-MS for determination of plasma glycerol enrichment
(13). Breath 13CO2 enrichment was
measured by isotope ratio-mass spectrometry (Metabolic Solutions,
Nashua, NH) (38).
Calculations.
Net skeletal muscle protein balance (synthesis-breakdown) was
determined by dilution and enrichment of phenylalanine across the
hindlimb as described by Gelfand and Barrett (16). Because phenylalanine is neither synthesized nor metabolized by skeletal muscle, the appearance rate (Ra) of unlabeled phenylalanine
reflects muscle protein breakdown, whereas the rate of disappearance
(Rd) of labeled phenylalanine estimates muscle protein
synthesis (16). Phenylalanine Rd was calculated by
multiplying the fractional extraction of the labeled phenylalanine
(based on plasma arterial and venous phenylalanine enrichments and
concentrations) by the arterial phenylalanine concentration and leg
plasma flow (16, 47). Net phenylalanine Ra was
calculated by subtracting the net arteriovenous balance of
phenylalanine across the hindlimb from the phenylalanine Rd
(16, 47). Rates of skeletal muscle protein breakdown and net
synthesis were determined from the phenylalanine Rd and
Ra, with the assumption that 3.8% of skeletal muscle
protein is comprised of phenylalanine.
Steady-state rates of whole body glucose disappearance (Rd)
were calculated by dividing the
D-[6,6-2H2]glucose infusion rate
by the plasma [2H2]glucose enrichment
(47). With this method, the deuterium label is lost during
the phosphoenolpyruvate cycle, is diluted into the total
body water pool, and is not recycled. The steady-state rates of whole
body leucine appearance (Ra; an estimate of whole body
protein breakdown) were calculated by dividing the
[13C]leucine infusion rate by the plasma
[13C]KIC enrichment (47). Plasma KIC
provides a better estimate of intracellular leucine enrichment than
does plasma leucine enrichment because KIC is derived from
intracellular leucine metabolism (47). The endogenous
leucine Ra was calculated by subtracting the exogenous leucine Ra from the total leucine Ra. Exogenous
leucine Ra from the nutrient supplement was calculated with
the assumption that the protein consumed was 7.8% leucine and that the
plasma leucine Ra from the exogenous supplement was
constant (33.03 µmol/min) over the entire 3-h period in both EARLY
and LATE. This assumption was validated by two lines of evidence.
First, when 100 mg of L-[2H8]valine were administered
with the supplement, arterial
L-[2H8]valine enrichments were
similar at corresponding time points in both EARLY and LATE (mean = 0.19 ± 0.01 vs. 0.19 ± 0.01%). A second line of evidence
deals with the concentration of hormones and metabolites. For example,
plasma leucine increased within 30 min after supplement intake and
remained similarly elevated for 3 h after intake in both EARLY and
LATE (Fig. 2). Breath
13CO2 production was determined by multiplying
the total CO2 production rate by the breath
13CO2 enrichment (47). The rate of
whole body leucine oxidation was calculated by dividing breath
13CO2 production by 0.8 (correction factor for
the retention of 13CO2 in the bicarbonate pool)
(2) and by the plasma KIC enrichment. The nonoxidative
leucine Rd, an estimate of whole body protein synthesis,
was determined indirectly by subtracting leucine oxidation from total
leucine Ra. Rates of whole body protein breakdown, amino
acid oxidation, and protein synthesis were calculated from the
endogenous leucine Ra, the leucine oxidation rate, and the nonoxidative leucine Rd, respectively, assuming that 7.8%
of whole body protein is comprised of leucine (15). Because
glycerol released during lipolysis cannot be reincorporated into
triacylglycerol in the adipose cell because of the lack of glycerol
kinase activity, the Ra of endogenous glycerol multiplied
by 3 was used to determine rates of whole body lipolysis
(13). The endogenous glycerol Ra was
calculated by dividing the [2H5]glycerol
infusion rate by the plasma glycerol enrichment and subtracting the
exogenous glycerol infusion rate (47).

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Fig. 2.
Point-by-point timing of arterial enrichment of valine
and arterial concentrations of glucose, leucine, and essential amino
acids for the 3 h after postexercise nutrient ingestion. MPE, mole
percent excess.
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Rates of whole body amino acid, carbohydrate, and lipid oxidation were
determined from indirect calorimetry in combination with the leucine
oxidation data. The energy expended due to amino acid oxidation was
subtracted from the total EE, and the net rates of carbohydrate and
lipid oxidation were calculated on the basis of the nonprotein
respiratory quotient (23). The assumptions and limitations
of calculating net substrate oxidation on the basis of indirect
calorimetry measurements have been reviewed previously
(23). Net whole body nutrient balances were calculated by
subtracting whole body nutrient oxidation from nutrient intake.
Statistical analysis.
For each protocol, mean variables for each period (basal and the 3-h
postexercise intake periods) were calculated. Values presented in the
text and Figs. 1-7 are means ± SE for each period. No
differences were noted between treatments during the basal periods, and
thus the data for this period will be reported as the overall
means ± SE. Because each subject was studied twice, differences
between the mean values obtained during the postnutrient intake periods
were assessed using a repeated-measures analysis of variance with a
model of treatment within gender (Statistical Analysis System for
Windows, 1996, Release 6.12, SAS Institute, Cary, NC). A P
value <0.05 was required to reject the null hypothesis of no
difference between the means. Because gender groups responded to
treatments similarly, data are reported as means of all subjects combined.

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Fig. 3.
Net leg glucose uptake for 10 subjects given an
oral nutrient supplement either immediately after exercise (EARLY) or
3 h postexercise (LATE). *Significant difference (P < 0.05), EARLY vs. LATE.
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Fig. 4.
Rates of leg protein dynamics for 10 subjects given an
oral nutrient supplement either immediately after exercise (EARLY) or
3 h postexercise (LATE). *Significantly different (P < 0.05), EARLY vs. LATE.
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Fig. 5.
Rates of whole body protein dynamics for 10 subjects
given an oral nutrient supplement either immediately after exercise
(EARLY) or 3 h postexercise (LATE). *Significantly different
(P < 0.05), (EARLY) vs. (LATE).
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Fig. 6.
Point-by-point timing of leg and whole body glucose uptakes for 10 subjects given an oral nutrient supplement either immediately after
exercise (EARLY) or 3 h postexercise (LATE). *Significantly
different (P < 0.05), EARLY vs. LATE. Shaded bar represents
basal means ± SE.
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Fig. 7.
Point-by-point timing of leg and whole body protein synthesis for
10 subjects given an oral nutrient supplement either immediately after
exercise (EARLY) or 3 h postexercise (LATE). *Significantly
different (P < 0.05), EARLY vs. LATE. Shaded bar, basal
means ± SE.
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RESULTS |
Subject characteristics.
The ten healthy adult subjects (5 females and 5 males) were 20-41
yr of age and within 25% of ideal body weight (Table
1). Subjects were recreational athletes
with an average
O2 max of 33.2 ± 4.0 and 44.9 ± 2.9 ml · kg
1 · min
1 for females
and males, respectively. Whereas body mass index (BMI) was similar
between females and males, average body fat was 30.5 ± 3.4 and
17.0 ± 2.5% for females and males, respectively.
Metabolite and hormone concentrations.
Circulating glucose, lactate, and glycerol concentrations were not
different whether the nutrient supplement was given immediately after
exercise (EARLY) or 3 h after exercise (LATE; Table
2). In addition, the concentrations of
plasma insulin, glucagon, growth hormone, epinephrine, and
norepinephrine did not differ between EARLY and LATE. In contrast,
plasma cortisol concentration was 18% greater during EARLY than during
LATE.
Whereas amino acid concentrations increased after the supplement was
given in both instances, the concentrations of the individual plasma
amino acids were unaffected by the timing of the intake of the nutrient
supplement containing protein, with the exception of glutamine. In the
case of glutamine, the concentration was 19% greater when the
supplement was given EARLY vs. LATE. In addition, although the
plasma concentrations of BCAA and EAA were not different between EARLY
and LATE, the significant difference in glutamine concentrations causes
similar differences in NEAA and TAA between EARLY and LATE (Table
3).
Net glucose and amino acid balance across the leg.
The mean leg blood flow measurements
(ml · min
1 · 100 ml
1)
during basal (6.1 ± 0.8), EARLY (6.0 ± 0.9), or LATE
(5.2 ± 0.4) were not significantly different. Net leg glucose
uptake was 32.5 µg · min
1 · 100 ml
1 during the basal period. When the oral postexercise
supplement was given 3 h after the completion of exercise (LATE),
there was not a significant increase from basal for net leg glucose
uptake (Fig. 3). However, when the
supplement was given immediately after exercise (EARLY), net leg
glucose uptake was 3.5 times greater than basal or LATE.
During the basal period, there was a net uptake of aspartate,
glutamate, hydroxyproline, ornithine, serine, and taurine by the leg
and a net release of BCAA, EAA, NEAA, and TAA (Table
4). Net balance was significantly more
positive during EARLY, compared with LATE, for isoleucine, leucine,
lysine, phenylalanine, proline, valine, BCAA, EAA, NEAA, and TAA.
Therefore, as a result of these changes in individual amino acid
balance, there was a net leg uptake of BCAA, EAA, NEAA, and TAA with
EARLY, which contrasted with a net release of BCAA, EAA, NEAA, and TAA
in LATE.
Leg fractional extraction of phenylalanine also was significantly
affected by the timing of the oral postexercise nutrient supplement.
The mean leg fractional extraction of phenylalanine during the basal
period was 9.8 ± 1.1%. When the oral postexercise supplement was
given EARLY, mean leg fractional extraction of phenylalanine was
increased to 17.0 ± 2.4%. However, when the supplement was
administered 3 h after exercise, mean leg fractional extraction of
phenylalanine was only 7.6 ± 1.4%.
Leg protein dynamics.
Leg protein synthesis and net protein balance were both significantly
affected by the timing of an oral postexercise nutrient supplement
(Fig. 4). Leg protein synthesis was more
than 3 times greater for EARLY compared with LATE. Leg protein
breakdown, however, was not significantly different between the two
supplemental periods. Thus there was a net gain of protein in the leg
for EARLY but a net loss of leg protein for LATE, with an absolute
difference of 84.3 µg · min
1 · 100 mg
1 (+60.5 vs.
23.8
µg · min
1 · 100 ml
1, respectively).
Whole body protein dynamics.
Whole body proteolysis, as estimated by endogenous leucine
Ra, was not significantly affected by the timing of a
postexercise nutrient supplement (2.52 ± 0.17 mg · kg
1 · min
1 EARLY vs.
2.33 ± 0.15 mg · kg
1 · min
1 LATE; Fig.
5). However, whole body protein synthesis
was 12% greater (2.69 ± 0.13 vs. 2.40 ± 0.11 mg · kg
1 · min
1) for EARLY
vs. LATE. Thus there was a net whole body release of protein in the
postabsorptive basal period, but there was a net uptake (protein
balance was positive) when an oral nutrient supplement was given after
exercise, regardless of timing. Net whole body protein deposition was
significantly greater (0.17 ± 0.15 vs. 0.07 ± 0.07 mg · kg
1 · min
1) when the
oral postexercise supplement was given immediately after exercise vs.
3 h later.
Whole body glucose and glycerol kinetics.
Whole body glucose utilization was 1.97 ± 0.07 mg · kg
1 · min
1 during the
basal period. Whole body glucose utilization was 44% greater when the
oral postexercise supplement was given immediately after exercise vs.
3 h later (2.40 ± 0.11 mg · kg
1 · min
1 EARLY vs.
1.67 ± 0.12 mg · kg
1 · min
1 LATE).
Whole body glycerol flux (7.82 ± 1.18 µmol · kg
fat
1 · min
1 EARLY vs. 6.16 ± 1.04 µmol · kg
fat
1 · min
1 LATE) was unaffected by
the timing of the oral supplement postexercise.
Energy metabolism.
There was no change in total EE, normalized to body weight, in
EARLY vs. LATE (Table 5). In addition,
the proportion of energy derived from protein, carbohydrate, or lipid
was not different between the treatments. Whole body amino acid,
carbohydrate, and fat oxidation were also unaffected by the timing of
the oral postexercise supplement intake.
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DISCUSSION |
Although several studies have focused on the timing of
carbohydrate administration after exercise (21, 22, 43),
little is known concerning the timing of postexercise nutrient
supplementation and protein homeostasis. Therefore, the focus of the
present study was to examine whether the timing of postexercise
nutrient ingestion has an impact on whole body and leg protein
homeostasis in healthy adults. Each subject in the present study was
tested twice, with the nutrient supplement (10 g protein, 8 g
carbohydrate, 3 g fat) being consumed either immediately (EARLY)
or 3 h (LATE) after exercise. Whether taken immediately or 3 h after exercise, consumption of this supplement resulted in similar
plasma glucose and insulin concentrations in the plasma for the 3 h after ingestion. Furthermore, concentrations of all EAA and NEAA,
with the exception of glutamine, were similar during EARLY and LATE
periods. Leg blood flow and all other hormones and metabolites
measured, with the exception of cortisol, were also similar between the
two postexercise periods. Although the substrate and hormonal milieu
were similar whether the supplement was given immediately or 3 h
after exercise, the leg uptake of glucose and amino acids was greater
when the supplement was given immediately after exercise.
With the greater net uptake of amino acids and glucose during EARLY,
more substrate and energy were available within the leg for protein
synthesis. Although leg proteolysis was not significantly different
between the two treatments, leg protein synthesis was increased more
than threefold for EARLY vs. LATE. Hence, there was a net accretion of
leg protein when nutrients were ingested immediately after exercise,
which was in contrast to the net loss of leg protein when nutrients
were given 3 h after exercise. These results confirm the
conclusions of Okamura et al. (32). In that study, a 2-h
intraportal infusion of glucose and amino acids was given to canines
either immediately or 2 h after treadmill exercise. The earlier
nutrient infusion increased leg protein synthesis by ~35% but did
not alter leg proteolysis. In a longer-term study (10 wk), exercising
rats were fed a mixed meal either immediately or 4 h after
exercise (42). The rats that were fed immediately after
exercise for 10 wk had increased total hindlimb muscle weight and
decreased perirenal, epididymal, and mesenteric adipose tissue weight.
These findings support the more acute studies, such as the present
study and that of Okamura et al.
Whole body protein dynamics in the present study followed the same
pattern as leg protein dynamics, with rates of proteolysis being
similar but rates of protein synthesis and net protein deposition both
being greater during EARLY vs. LATE. Previously, it has been demonstrated that gastrointestinal tract tissue protein homeostasis is
in a net catabolic state, with protein breakdown increased by
40-50% during exercise (19, 46). Therefore, these
tissues also could potentially benefit from an environment that would promote net protein synthesis. However, it is not possible in this
study to speculate on changes in protein synthesis for nonexercising muscle tissues or specific nonskeletal muscle tissues (e.g., heart, kidney, or liver vs. intestinal tract).
The timing of carbohydrate administration after exercise has been
studied extensively in recent years. Ivy et al. (22)
demonstrated that a 25% glucose polymer supplement given immediately
postexercise dramatically increased rates of glycogenesis, but the rate
of glycogen storage was markedly decreased if ingestion was delayed for
2 h postexercise. The results from the present study confirm these
previous conclusions, because leg glucose uptake and whole body glucose
utilization were considerably greater when the nutrient supplement was
given immediately postexercise. Glycogen in exercised muscle can be
replenished within 24 h after exercise with the consumption of a
high (500-600 g) carbohydrate diet (4, 8). Glycogen
resynthesis is maximized at 5-8 mmol · kg wet weight of
muscle
1 · h
1 by providing
0.7-1.5 g of glucose per kg body weight every 2 h for up to
6 h after exhaustive exercise (14, 21). Furthermore, if a high-carbohydrate diet is continued for 3 days, muscle glycogen content rises above preexercise levels (40). These
observations have fostered the concept referred to as "carbohydrate
loading".
Whereas exercise increases lipoprotein lipase activity in muscle but
not in adipose tissue (39), plasma triacylglycerol concentrations are altered depending on the timing of sucrose consumption relative to exercise (43). The effects of when
a postexercise nutrient supplement is ingested on lipid metabolism in
rats also were reported by Suzuki et al. (42). In their
study, exercising rats were given a meal either immediately or 4 h
after exercise, and lipoprotein lipase activity in the soleus was 70% greater in the group given a meal immediately after exercise
(42). Thus more energy after exercise would be directed
away from fat stores and toward muscle stores, thereby potentially
facilitating muscle protein and glycogen synthesis.
Studies examining the timing of postexercise nutrient supplementation
suggest that repletion of leg nutrient stores is not determined by
nutrient intake alone. Insulin action also appears to play an important
role in controlling postexercise nutrient repletion. Insulin decreases
circulating concentrations of glucose, amino acids, and lipids,
promotes inward cellular transport of glucose and amino acids, enhances
glycogen synthesis, stimulates adipose cells to synthesize and store
fat, and promotes protein accretion (12). Although
circulating insulin is reduced during exercise, muscle glucose
transport (11, 18) and utilization (44) are
still enhanced with exercise as a result of improved insulin
sensitivity and responsiveness. Therefore, the role of exercise-stimulated insulin action may be critical to the observation that the rate of glycogen resynthesis is greater the earlier that carbohydrate intake is given after exercise. Although the exact time
course of this enhanced insulin action has not been defined, Burstein
et al. (7) demonstrated that a majority of this effect is
lost after 60 h. The present study suggests that the peak
stimulation in whole body glucose utilization and leg glucose uptake
occurred within the 1st h after immediate postexercise consumption of
the nutrient supplement (Fig. 6). These
values steadily decreased to near basal over the subsequent 3 h.
Interestingly, this response occurred during the first 3 h after
exercise despite a lingering elevation in circulating cortisol, which
is known to blunt the response of carbohydrate metabolism to insulin
(36). When the same supplement was administered 3 h
postexercise, there was no change in these measures of carbohydrate
utilization compared with basal, suggesting that peak sensitivity to
insulin-mediated carbohydrate metabolism occurs very soon after exercise.
Insulin also has been shown to be central in the regulation of protein
dynamics (12). Numerous reports exist demonstrating that
stimulation of amino acid transport, promotion of whole body and muscle
protein synthesis, and inhibition of proteolysis occur when amino acid
availability and insulin concentrations are increased (12). Very little is known, however, regarding how
exercise modulates insulin's effects on protein metabolism. In the
present study, whole body and leg protein synthesis were increased
during the same postexercise period that insulin responsiveness to
glucose metabolism appeared to be increased. Furthermore, although the changes were not as large as those noted for glucose utilization, protein synthesis also was greatest immediately after exercise and
tended to decrease over the 3-h period (Fig.
7). Also similar to carbohydrate
metabolism, there was no change in whole body or leg protein synthesis
when the same supplement was provided 3 h after exercise. These
data suggest that exercise's modulation of insulin action also may
play a central role in the regulation of protein synthesis. This
conclusion is supported by a previous report with canines in which net
muscle protein balance became positive within 15 min after initiation
of a postexercise glucose-amino acid infusion and continued as such for
the entire 2-h infusion (32). In addition, when the
nutrient supplementation was discontinued in the canines, net muscle
protein balance became negative again.
Insulin also is important in the regulation of amino acid transport
(12). Insulin-stimulated amino acid transport was elevated by muscular contraction even in the absence of an increase in insulin
binding (48). Although transport was not directly measured in the present study, fractional extraction of phenylalanine by the leg
was twofold greater for EARLY vs. LATE supplementation, suggesting that
leg tissues were more effective at removing the amino acids presented
to them immediately vs. 3 h after exercise. Taken together with
the information on protein synthesis, these data support enhanced
insulin sensitivity as a central component of the mechanism involved in
how the timing of nutrient supplementation after exercise alters
utilization of a nutrient load.
The importance of replenishing muscle glycogen content for subsequent
moderate- to heavy-intensity exercise is also well established (3, 33, 41). Inadequate muscle glycogen results in fatigue and inability to train at high intensities. Therefore, sufficient stores of muscle glycogen are essential for optimum performance during
intense, prolonged exercise. Although muscle glycogen was not tested in
this study, it is reasonable to speculate that similar importance can
be placed on the repair and synthesis of muscle protein after exercise.
Muscle glycogen resynthesis after prolonged exercise has been
investigated with respect to amount, type, physical form, and timing of
the ingested carbohydrate (45). Similar interactions with
these variables and postexercise protein synthesis have not been well
documented. Factors that can potentially affect nutrient utilization
include age, gender, body composition, and physical training of the
participants; the type, duration, and intensity of exercise performed;
the amount and physical form of the nutrient ingested; and the protein
and energy intake in the days before exercise. Extreme care was taken
to maintain consistency in these variables between the two treatments
in this study. However, it is possible that alterations in these
variables from those used in the present study may result in different
conclusions. For example, a more intense exercise may result in more
prolonged catabolic hormone concentrations, thereby blunting insulin
action. Furthermore, the ingestion of a supplement consisting of free amino acids, as opposed to the ingestion of an intact protein source,
may produce significantly different results.
Therefore, our study clearly indicates that the timing of postexercise
nutrient supplementation has a significant impact on whole body and leg
protein homeostasis, similar to that observed for carbohydrate
homeostasis. Thus whole body and leg protein synthesis, as well as net
protein deposition, is enhanced when nutrients are consumed immediately
after exercise as opposed to 3 h later. These data suggest that
exercise's modulation of insulin action may impact whole body and
muscle protein accretion, as well as glucose deposition.
 |
ACKNOWLEDGEMENTS |
The aid of the personnel and use of the facilities at the General
Clinical Research Center (RR-00095), Clinical Nutrition Research Unit
(DK-26657), and Diabetes Research and Training Center (DK-20593) at
Vanderbilt University is greatly appreciated. In addition, we thank the
Pharmavite Corporation (Mission Hills, CA) for their support. We
appreciate the outstanding technical expertise of the personnel in the
Cardiac Catheterization Laboratory at Vanderbilt University Medical
Center in completing this research, and we are grateful for the expert
technical and analytical assistance of Suzan Vaughan, Mu Zheng, Donna
Schot, and Wanda Snead. Finally, we thank the subjects for their loyal
compliance and participation.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: P. J. Flakoll, Dept. of Surgery, CC-2306 Medical Center North, Vanderbilt Univ. Medical Center, Nashville, TN 37232-2733 (E-mail:
Paul.Flakoll{at}mcmail.vanderbilt.edu).
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.
Received 30 August 2000; accepted in final form 14 February 2001.
 |
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