Vol. 93, Issue 5, 1797-1805, November 2002
Partial restoration of dietary fat induced metabolic
adaptations to training by 7 days of carbohydrate diet
Jørn W.
Helge1,
Peter
W.
Watt2,
Erik A.
Richter1,
Michael J.
Rennie2, and
Bente
Kiens1
1 Copenhagen Muscle Research Centre, Department of
Human Physiology, University of Copenhagen, DK-2100 Copenhagen Ø,
Denmark; and 2 Division of Molecular
Physiology, University of Dundee, Dundee DD1 4HN,
Scotland
 |
ABSTRACT |
We tested the hypothesis
that a shift to carbohydrate diet after prolonged adaptation to fat
diet would lead to decreased glucose uptake and impaired muscle
glycogen breakdown during exercise compared with ingestion of a
carbohydrate diet all along. We studied 13 untrained men; 7 consumed a
high-fat (Fat-CHO; 62% fat, 21% carbohydrate) and 6 a
high-carbohydrate diet (CHO; 20% fat, 65% carbohydrate) for 7 wk, and
thereafter both groups consumed the carbohydrate diet for an eighth
week. Training was performed throughout. After 8 wk, during 60 min of
exercise (71 ± 1% pretraining maximal oxygen uptake) average leg
glucose uptake (1.00 ± 0.07 vs. 1.55 ± 0.21 mmol/min) was
lower (P < 0.05) in Fat-CHO than in CHO. The rate of
muscle glycogen breakdown was similar (4.4 ± 0.5 vs. 4.2 ± 0.7 mmol · min
1 · kg
dry wt1) despite a significantly higher preexercise
glycogen concentration (872 ± 59 vs. 688 ± 43 mmol/kg dry
wt) in Fat-CHO than in CHO. In conclusion, shift to carbohydrate diet
after prolonged adaptation to fat diet and training causes increased
resting muscle glycogen levels but impaired leg glucose uptake and
similar muscle glycogen breakdown, despite higher resting levels,
compared with when the carbohydrate diet is consumed throughout training.
fat diet; [13C]palmitate; arteriovenous
balance; carbohydrate loading
| |
INTRODUCTION |
DIETARY INFLUENCE ON
MUSCLE metabolism during exercise has been studied since the
1930s when Christensen and Hansen (7) observed that the
preexercise diet was of major importance for substrate metabolism as
well as endurance during exercise. We have previously shown (16,
17) that adaptation to a fat-rich diet during physical training
for 7 wk increases utilization of lipids (16, 17) and
decreased rate of muscle glycogen breakdown during submaximal exercise
(17) compared with a group of similar subjects training on
a carbohydrate-rich diet (16, 17). Interestingly, a prior
study demonstrated (16) that, despite very high
glycogen stores, induced by consuming a carbohydrate-rich diet after
prolonged fat diet adaptation, the subjects were unable to utilize the
glycogen stores efficiently, and exhaustion during exercise occurred
despite very high residual muscle glycogen stores of ~500 µmol/g
dry wt. Thus prolonged fat adaptation seems to induce an inability to efficiently use muscle glycogen during exercise, and this adaptation is
not overcome by 1 wk of carbohydrate diet. However, we did observe that
toward the end of exercise the venous plasma glucose concentration in
contrast to what might have been expected was higher in the group
switching from fat to carbohydrate for the last week of training than
in the group ingesting a carbohydrate-rich diet all along and from this
we speculated that glucose utilization was attenuated. One goal of the
present study therefore was to investigate whether glucose uptake may
be attenuated under such circumstances.
In a recent study, Burke and colleagues (5) demonstrated
that 5 days of fat adaptation followed by 1 day of carbohydrate-rich diet resulted in muscle glycogen sparing and a higher fat oxidation during submaximal exercise compared with subjects continuously consuming a carbohydrate-rich diet. In contrast, neither a higher fat
oxidation nor a difference in muscle glycogen breakdown rate was
observed in our laboratory's previous study after a dietary switch to
a carbohydrate-rich diet for 1 wk after 7 wk on a fat-rich diet,
compared with subjects consuming a carbohydrate-rich diet all along
(16). Still, because higher preexercise muscle glycogen stores in the group switching from fat to carbohydrate diet by itself
would be expected to increase muscle glycogen breakdown (15), it could be argued that identical muscle glycogen
breakdown rates in the two groups in fact represent a relative
impairment of breakdown after the dietary switch. However, in neither
of these latter studies was a detailed characterization of muscle metabolism during exercise made.
The aim of the present study was to investigate substrate metabolism
after prolonged adaptation to training and a fat-rich diet followed by
a carbohydrate-rich diet and to compare muscle metabolism to that
observed when a carbohydrate-rich diet was taken throughout the period.
| |
METHODS AND MATERIALS |
Subjects.
Thirteen healthy, untrained male subjects, age 27 ± 1 yr, height
182 ± 2 cm, weight 87 ± 3 kg and maximal oxygen uptake
3.9 ± 1 liters O2/min participated in the study. The
fiber type composition in the vastus lateralis muscle was 55 ± 3% type I, 34 ± 2% type IIA, and 11 ± 2% type IIX fibers
(type IIB in old nomenclature). Subjects were fully informed of the
nature and the possible risks associated with the study before they
volunteered to participate. The study was approved by the Copenhagen
Ethics Committee, and all subjects gave written consent.
Design.
The experiment was a longitudinal diet-training intervention study.
Initially, subjects were randomized into two groups. Over 8 wk, both
groups followed a training program. Over the first 7 wk, one group
consumed a fat-rich diet and the other group a carbohydrate-rich diet
(see below for details). During the last week, both groups consumed a
carbohydrate-rich diet. Through the remainder of this paper, the group
that switched diet will be referred to as Fat-Carbohydrate (Fat-CHO in
tables and figures) and the other group as Carbohydrate (CHO in tables
and figures). After 8 wk of the diet and training regimen, substrate
metabolism was investigated in a 60-min exercise bout performed at
~70% of maximal oxygen uptake on a modified Krogh bicycle ergometer.
Over the experimental period, the subject's maximal oxygen uptake was determined before diet change or training, after 3.5 and 6.5 wk of the
experiment. An exercise test was also performed after 7 wk, the results
of which have been published separately (17).
Experimental diets.
The experimental diets are similar to those applied and described in
our laboratory's previous studies (16). In brief, the energy composition of the fat-rich diet was 21% carbohydrate, 17%
protein, and 62% fat, and the carbohydrate-rich diet was 65% carbohydrate, 15% protein, and 20% fat. Thus the diets had a markedly different fat and carbohydrate content but a similar protein content. The habitual diet and energy intake were determined from 4-day diet
records, and, in addition, individual energy intakes were estimated
from the World Health Organization's equation for calculation of
energy needs (34). On the days of training, the calculated energy expenditure during training was added to the daily energy intake
and consumed immediately after training. During the experimental period
all food intake was strictly controlled and weighed to within 1 g.
The subjects weighed themselves every morning, and the individual
dietary energy intake was adjusted such that body weight changes were minimized.
Materials.
[1-13C]palmitate (99% enriched) and
NaH13CO3 were purchased from Tracer
Technologies (Newton, MA). The palmitic acid tracer in solution was
added to methanolic potassium hydroxide to form the potassium salt,
which was then dried under nitrogen, redissolved in sterile water, and
passed through a 0.22-µm sterile filter. It was then mixed with
sterile 20% (wt/vol) human albumin (State Serum Institute, Copenhagen,
Denmark), to which it was bound.
Experimental protocol.
Subjects were asked to refrain from physical activity 2 days before the
start of the studies. The subjects reported to the laboratory in the
morning after a 12-h fast after traveling to the laboratories either by
car or bus. After subjects spent 30 min in a supine position, a
needle biopsy was taken with suction from the vastus lateralis
muscle by using local anesthesia with 5 ml of 1% lidocaine
(3). After this, the training and diet regimen was begun.
Over 8 wk, both dietary groups followed an identical, supervised
training program using a bicycle ergometer. During the whole period,
physical training was performed four times a week. Each training
session lasted between 60 and 75 min, and exercise intensity, which was
carefully controlled, varied from 50 to 85% of maximal oxygen uptake.
The training program consisted of four different protocols with
exercise intervals varying in length and duration interspersed with
breaks of active recovery. Training intensity was adjusted to changes
in maximal oxygen uptake measured after 3.5 wk of the training period.
At every training session, heart rate was monitored; pulmonary oxygen uptake was measured frequently. Thus the training regime could be
monitored and adapted as required.
After 7 wk, all subjects arrived overnight fasted, and a muscle biopsy
was obtained from the vastus lateralis for substrate concentration
measurement, described in more detail in a separate paper
(17). However, these muscle substrate data are also
presented here. After the muscle biopsy, the subjects
participated in an exercise test (17). At this stage, the
subjects on the fat diet switched to the carbohydrate-rich diet, and
the group that had consumed the carbohydrate-rich diet continued on
that diet. Both groups continued the training over the ensuing week.
After a week, the subjects again reported to the laboratory, overnight
fasted and having not trained for 2 days. After 30 min of rest in the supine position, Teflon catheters were placed by an aseptic technique in the femoral artery and vein of the same leg under local anesthesia (1% lidocaine), and the tips were advanced to ~2 cm above (arteries) and below (vein) the inguinal ligament. A thermistor (Edslab Probe 94-030-2.5-F, Baxter Healthcare) for measuring venous blood temperature was advanced 8 cm proximal to the catheter tip. A catheter was also
inserted into an antecubital vein for the infusion of
[1-13C]palmitate. The catheters were flushed with sterile
sodium citrate (~3.6 mM). A needle biopsy was obtained with suction
from the vastus lateralis muscle. Subjects were then placed in a
semisupine position, where they rested for 30 min. Blood was sampled
simultaneously from the femoral artery and vein, and femoral venous
blood flow was measured by the thermodilution method by use of bolus
injections of 3 ml ice-cold saline into the femoral vein
(1). Resting oxygen uptake and background breath
enrichment samples were then collected. Subsequently, the bicarbonate
pool was primed with a bolus of NaH13CO3 (0.1 mg/kg), and a continuous infusion of [1-13C]palmitate
(99% enriched) was started by use of a calibrated syringe pump (Vial
Medical SE 200B, Simonsen & Weel, Copenhagen, Denmark) set at a
constant rate of 0.06 µmol · kg1 · min1.
Blood samples were taken after 50, 55, and 60 min of infusion. Subjects
were then positioned on the Krogh cycle ergometer, and exercise was
begun at ~80% of their individual pretraining maximal oxygen uptake
without a prior warm-up. During exercise, blood samples were taken
every 15 min. For the analyses, very-low-density lipoprotein-triacylglycerol (VLDL-TG) concentration was only measured at 0, 30, and 60 min, and the hormone concentrations were not measured
in the 45-min sample. Venous blood flow was measured frequently via
continuous infusions of ice-cold saline according to the thermodilution
principle (1). Expired air was sampled into Douglas bags
at the same time, and subsequently small aliquots were collected into
evacuated glass tubes (Vacutainer, Becton Dickinson, Meylan, France)
for analysis of 13CO2 enrichment. Exercise
heart rate was recorded continuously with a PE 3000 Sports Tester
(Polar Electro). Water intake during exercise was standardized with
subjects drinking 200 ml of water every 20 min. Immediately after 60 min of exercise, a further biopsy was taken from the vastus lateralis
muscle of the opposite leg through a new incision, suitably anesthetized.
Analyses.
Fatty acids (FA) were extracted from plasma, isolated by thin-layer
chromatography, and converted to their methyl esters. The arterial and
venous isotopic enrichment of plasma [13C]palmitate was
determined by gas chromatography-mass spectrometry (GC-MS, INCOS XL,
Finnigan Mat, Hemel, Hempstead, UK) by selected ion monitoring of ions
at mass-to-charge ratio of 270 and 271. Heptadecanoate
(C17) was used as an internal standard for quantification of total palmitate. Enrichment of 13CO2 in
expired air was analyzed by isotope ratio-mass spectrometry (Europa
Scientific 2020 IRMS) as previously described (28). The
concentration of isotope in the infusate was determined, so that the
exact infusion rate could be calculated.
Blood glucose and lactate were analyzed on a glucose and lactate
analyzer (Yellow Springs Instruments, Yellow Springs, OH). Plasma
glycerol was analyzed as described by Wieland (33). Total plasma FA was measured fluorometrically as described by Kiens et al.
(19). VLDL-TG in serum was isolated by ultracentrifugation at a density of 1.006 g/cm3 and then analyzed as
described by Kiens et al. (20). Insulin in arterial plasma
was determined by use of a radioimmunoassay kit (Insulin RIA100,
Pharmacia, Sweden), and catecholamines in arterial plasma were
determined by a radio enzymatic procedure (8). Blood
oxygen saturation was measured on an OSM-2 hemoximeter (Radiometer
Copenhagen). Hemoglobin was determined spectrophotometrically on the
hemoximeter by the Drabkin and Austin cyan-methemoglobin method
(11a). Blood gases (PCO2,
PO2) and pH were measured with the Astrup
technique (ABL 30, Radiometer, Copenhagen, Denmark). Hematocrit was
determined in triplicate from microcapillary tubes.
The biopsies were frozen in liquid nitrogen within 10-15 s of
sampling. Before freezing, a section of the samples was cut off,
mounted in embedding medium, and frozen in isopentane cooled to its
freezing point in liquid nitrogen. Both parts of the biopsy were stored
at 
80°C until further analysis. Before biochemical analysis, muscle
biopsy samples were freeze-dried and dissected free of connective
tissue, visible fat, and blood with the use of a stereomicroscope.
Muscle glycogen concentration was determined as glucose residues after
hydrolysis of the muscle sample in 1 M HCl at 100°C for 2 h
(22). Muscle triacylglycerol was determined as previously
described (21). In brief, 20 mg wet wt of muscle tissue
were freeze-dried and dissected free of all visible adipose tissue,
connective tissue, and blood by the use of a stereomicroscope, leaving
the muscle fibers for further analysis. The muscle fibers were mixed,
and ~1 mg dry wt of the pooled fibers was used for measurement of the
intramyocellular triacylglycerol concentration. Glycerol from the
degraded triacylglycerol was assayed fluorometrically as described
previously (21). Serial transverse muscle sections were
stained for myofibrillar ATPase to identify fiber type composition (4). Total muscle GLUT-4 protein content was assayed by
Western blotting using a primary antibody against the 13 COOH-terminal amino acids of GLUT-4.
Whole body oxygen uptake and carbon dioxide excretion at rest and
during exercise were determined by collection of expired air in Douglas
bags. The volume of air was measured in a Collins bell-spirometer
(Tissot principle, Collins W.E., Braintree, MA), and the fractions of
oxygen and carbon dioxide were determined with paramagnetic (Servomex)
and infrared (Beckmann LB-2) systems, respectively. Gases of known
composition were used to calibrate each system regularly.
Calculations.
Uptake and release of substrates and metabolites across the leg were
calculated from femoral arterial and venous differences multiplied by
plasma or blood flow, according to Fick's principle. Indirect
calorimetry calculations were performed according to the stoichiometric
equations given by Frayn (12). Substrate balance across
the leg was calculated using an active muscle mass of 4.6 kg for each
leg, which was estimated as the difference between total carbohydrate
oxidation and measured glucose uptake and lactate release divided by
the measured muscle glycogen breakdown. To assess the contribution of
protein oxidation to exercise, a nitrogen excretion rate of 135 µg · kg1 · min1
was used, as described by Romijn et al. (27). FA oxidation was determined by converting the rate of triacylglycerol oxidation (in
g · kg1 · min1)
to its molar equivalent, with an average molecular weight of triacylglycerol assumed to be 860 g/mol (12).
The calculations of rate of appearance (Ra) and
disappearance (Rd), were performed by the use of Stele's
non-steady-state equations (31) modified for the use of
stable isotope tracer infusion (9, 27)
where F is infusion rate,
F13CO2 is the content of
13CO2 in the breath, V the effective volume of
distribution (40 ml/kg), Cpalmt1 and
Cpalmt2 are the concentrations of
plasma palmitate at time t1 and
t2, and Et1 and
Et2 are the palmitate enrichments,
respectively. The Ra of FAs was determined as the
product of the fractional contribution of palmitate to total FA
concentration and the Ra palmitate. The percentage of the
tracer infused that was oxidized was calculated as the
where C is the acetate correction factor (C = 0.9) as
reported by Sidossis and colleagues (29). In the present
experiment, it was not ethically feasible to do acetate correction
trials for each individual; thus we choose to apply an exercise
correction at 0.9 as described and utilized previously
(29).
The plasma FA oxidation was determined as
The estimated oxidation of FAs originating from sources other
than plasma FA was calculated as
Across-the-leg extraction can be calculated as
The calculated tracer-derived fractional extraction was used to
calculate actual tracer determined FA uptake over the leg as the
measured arterial FA delivery multiplied by the fractional extraction.
Statistics.
Results are given as means ± SE. Two-way ANOVA with repeated
measures for the time factor was performed to test for changes due to diet and/or time. In the case of significant main effects or
interactions, a Student-Newman-Keuls post hoc test was performed to
discern statistical differences. In all cases, an 
of 0.05 was taken
as the level of significance.
| |
RESULTS |
The habitual dietary energy and nutrient intake were similar in
the two groups (Table 1). Over the
experimental period, energy intake was also similar in the two groups
and significantly higher than the habitual daily energy intake.
Throughout the 8 wk, the subjects adhered to the prescribed dietary
intake both during the first 7 wk (17) and during the 8th
week, as evidenced by the close resemblance between the actual intakes
and the prescribed dietary contents (Table 1). After the switch from a
high-fat to a high-carbohydrate diet, the Fat-Carbohydrate group
increased (P < 0.05) their daily intake of
carbohydrates (in g) by 221% and vice versa decreased
(P < 0.05) their fat intake by 65%. Protein intake
was slightly lower (P < 0.05) in the Fat-Carbohydrate
group than in the Carbohydrate group. Over the experimental period body weight decreased (P < 0.05) similarly in both groups
from 87.4 ± 2.9 to 86.1 ± 2.9 kg.
View this table:
[in this window]
[in a new window]
|
Table 1.
Dietary content of the prescribed diets and the daily habitual and
experimental dietary energy and nutrient intake
|
|
Subjects trained under supervision a total of 31 ± 1 times.
Before the experimental period, maximal oxygen uptake was similar in
the two groups at 3.8 ± 0.1 and 4.1 ± 0.2 l/min, and after the training period it was similarly increased (P < 0.05) to 4.1 ± 0.1 and 4.4 ± 0.2 l/min in the
Fat-Carbohydrate group and in the Carbohydrate group, respectively.
After 8 wk, subjects exercised at an oxygen uptake of 2.9 ± 0.1 l/min, which was equivalent to a workload of 72 ± 3 and 70 ± 2% of posttraining maximal oxygen uptake in the Fat-Carbohydrate
group and in the Carbohydrate group, respectively. Over the first 15 min of exercise, heart rate increased (P < 0.05) from
rest to 155 ± 5 and 150 ± 2 beats/min, whereafter a
progressive increase (P < 0.05) to 165 ± 5 and
160 ± 4 beats/min was observed in the Fat-Carbohydrate and the
Carbohydrate groups, respectively. During exercise, respiratory
exchange ratio (RER) values remained constant throughout the exercise
period in both Fat-Carbohydrate (0.94 ± 0.01) and Carbohydrate
(0.92 ± 0.02) groups, and RER was similar between the
groups. During exercise, whole body fat oxidation (Table
2) and carbohydrate oxidation (175 ± 4 and 186 ± 5 µmol · min1 · kg1
in the Fat-Carbohydrate and the Carbohydrate group, respectively) were
similar between groups.
Blood samples.
Resting leg blood flow was similar in the two groups: 0.29 ± 0.04 and 0.36 ± 0.04 liters blood/min in the Fat-Carbohydrate group
and in the Carbohydrate group, respectively. The blood flow increased
(P < 0.05) similarly during the first 15 min of
exercise to 6.3 ± 0.3 and 6.0 ± 0.2 liters blood/min in the
Fat-Carbohydrate and Carbohydrate group, respectively, after which no
further increases were observed. In the Fat-Carbohydrate group,
arterial blood glucose concentrations increased (P < 0.05) progressively and continuously through exercise, whereas in the
Carbohydrate group the arterial glucose increased (P < 0.05) until 30 min, after which point it decreased (P < 0.05) at the termination of exercise down to the initial value (Fig.
1A). After 60 min, arterial
glucose concentration was borderline significantly higher
(P = 0.07) in the Fat-Carbohydrate group diet than in
the Carbohydrate group. Glucose delivery was not significantly
different between groups and was on average through exercise 30.2 ± 1.6 and 29.9 ± 1.4 mmol/min in the Fat-Carbohydrate group and
in the Carbohydrate group, respectively. In both groups, during the
exercise bout, glucose uptake across the leg increased similarly, and
at all time points the uptake was lower (P < 0.05) in
the Fat-Carbohydrate group than in the Carbohydrate group (Fig. 1B). Glucose clearance increased (P < 0.05)
across the exercise bout to 0.20 ± 0.02 l/min in the
Fat-Carbohydrate group and to 0.35 ± 0.06 l/min in the
Carbohydrate group, and during the later 30 min glucose clearance was
lower (P < 0.05) in the Fat-Carbohydrate group than in
the Carbohydrate group. Arterial blood lactate concentrations increased
(P < 0.05) similarly from rest to 15 min to 2.3 ± 0.4 mmol/l in both groups, and no further changes were observed.
Throughout the exercise, lactate release was similar between the
groups, and, after an initial increase to 0.72 ± 0.24 mmol/min
after 15 min (P < 0.05), a continuous decrease
(P < 0.05) was observed across the rest of the
exercise bout to 0.30 ± 0.11 mmol/min.
View larger version (13K):
[in this window]
[in a new window]
|
Fig. 1.
Arterial blood glucose concentrations (A) and
glucose uptake across the leg (B) at rest and during
exercise after 8 wk of training and adaptation to 7-wk fat-rich
followed by 1-wk high-carbohydrate diet (Fat-CHO) or 8 wk
carbohydrate-rich diet (CHO). Values are means ± SE.
* P < 0.05 compared with resting values;
 P < 0.05, Fat-CHO vs. CHO
|
|
Arterial plasma FA concentrations were not significantly different
during exercise in the two groups (Fig.
2A). After 15 min of exercise,
arterial plasma FA concentrations were at a nadir, after which a
continuous increase (P < 0.05) was observed until the
end of exercise in both groups (Fig. 2A). During the 60 min of exercise, FA delivery was not significantly different between groups, averaging 1.18 ± 0.24 and 1.52 ± 0.22 mmol/min in
the Fat-Carbohydrate group and the Carbohydrate group, respectively. During exercise, plasma net FA uptake across the leg was lower (P < 0.05) in the Fat-Carbohydrate group than in the
Carbohydrate group, averaging 99 ± 24 and 166 ± 28 µmol/min, respectively. The FA clearance was not significantly
different between groups and was on average 0.24 ± 0.07 l/min in
the Fat-Carbohydrate group and 0.34 ± 0.04 l/min in the
Carbohydrate group during exercise. During the first 30 min of
exercise, the average arterial serum VLDL-TG concentration was
1.06 ± 0.08 and 0.76 ± 0.15 mmol/l in the Fat-Carbohydrate
and Carbohydrate groups, respectively, and a significant decrease, to
0.96 ± 0.13 and 0.70 ± 0.29 mmol/l for Fat-Carbohydrate and
Carbohydrate, respectively, was observed at 60 min. No
measurable VLDL-TG-uptake was observed across the leg during the
exercise, 0.02 ± 0.07 and 0.02 ± 0.06 mmol/min in the
Fat-Carbohydrate group and in the Carbohydrate group, respectively. The
arterial plasma glycerol concentration was similar in the two groups
and increased (P < 0.05) continuously from 40 ± 4 µmol/l at rest to 136 ± 20 µmol/l at the end of
exercise. During exercise, plasma glycerol release across the
leg was similar between groups and increased (P < 0.05) from rest 17 ± 2 to 84 ± 23 µmol/min after 15 min.
After 30 min of exercise, a small decrease (P < 0.05)
was observed to 31 ± 17 µmol/min, whereafter plasma glycerol release was not further changed.
View larger version (15K):
[in this window]
[in a new window]
|
Fig. 2.
Arterial plasma fatty acid (FA) concentrations
(A) and total tracer-determined FA uptake across the leg
(B) during exercise after 8 wk of training and adaptation to
Fat-CHO or CHO diets. Values are means ± SE.
* P < 0.05 compared with rest.
|
|
The arterial epinephrine concentration increased
(P < 0.05) similarly from 0.71 ± 0.21 and
0.52 ± 0.09 nmol/l at rest to 1.79 ± 0.21 and 1.93 ± 0.41 nmol/l after 60 min in the Fat-Carbohydrate group and in the
Carbohydrate group, respectively. Likewise, the arterial norepinephrine
concentration increased (P < 0.05) similarly from
4.2 ± 1.6 and 3.7 ± 1.5 nmol/l at rest to 14.4 ± 1.3 and 15.8 ± 2.3 nmol/l after 60 min of exercise in
Fat-Carbohydrate and Carbohydrate, respectively. The arterial plasma
insulin concentration decreased (P < 0.05) from
10.7 ± 3.7 and 6.2 ± 1.1 µU/ml at rest to 6.8 ± 1.3 and 3.5 ± 0.4 µU/ml after 15 min of exercise in
Fat-Carbohydrate and Carbohydrate, respectively, after which no further
changes were observed. At rest, the plasma insulin concentration was
significantly higher in the Fat-Carbohydrate group than in the
Carbohydrate group; however, during exercise no significant difference
between groups was discernable.
Muscle samples.
Muscle glycogen concentration before training was similar in the two
groups (Table 3). After 7 wk, the
glycogen concentration was unchanged after fat diet adaptation and
significantly lower (P < 0.05) than after 7 wk of
carbohydrate diet, whereas after the 7-wk carbohydrate diet the
glycogen concentration was increased (P < 0.05) by
46% compared with the initial values (reported in Ref.
17). After 8 wk, the glycogen concentration was
significantly higher (P < 0.05) in the
Fat-Carbohydrate group than in the Carbohydrate group and higher
(P < 0.05) than the initial values (Table 3). In the
Carbohydrate group, glycogen storage remained at the level observed
after 7 wk. Muscle glycogen breakdown was similar in the
Fat-Carbohydrate group and in the Carbohydrate group across the 60 min
of exercise (Table 3). After 8 wk, muscle triacylglycerol concentrations were similar to the initial values in both groups, and
in the Fat-Carbohydrate group the values were significantly lower
(P < 0.05) than those reported after 7 wk (reported in
Ref. 17). This finding clearly implies that muscle
triacylglycerol stores are prone to rather large fluctuations when
nutrient composition and physical activity are markedly altered. The
total muscle GLUT-4 protein content was significantly increased
(P < 0.05) with training after 7 wk in both groups
(Table 3). After 8 wk, total muscle GLUT-4 protein content was not
further changed from the 7-wk values.
Stable isotopes and substrate kinetics.
The enrichment of plasma [13C]palmitate to
[12C]palmitate, the tracer-to-tracee ratio, decreased
(P < 0.05) from rest to 15 min into the exercise bout,
after which a stable plateau was maintained until the termination of
exercise (Table 2). During exercise, the arterial palmitate enrichment
was similar between groups, and, as expected, the arterial enrichments
were higher (P < 0.05) than the venous enrichments
during exercise. This resulted in an average fractional extraction over
the exercise calculated to 28 ± 14 and 22 ± 3% and a net
extraction of 9 ± 2 and 10 ± 1% in the Fat-Carbohydrate
group and the Carbohydrate group, respectively. The tracer-derived
total leg FA-uptake during exercise from 30 min and onward was not
different between groups (Fig. 2B). The release of FA,
calculated as the total leg FA uptake (tracer derived) minus the net FA
uptake, was similar between groups (157 ± 37 µmol/min
Fat-Carbohydrate; 155 ± 61 µmol/min Carbohydrate). The whole body Ra and Rd of palmitate and thus FA
was stable across the last 30 min of exercise in both groups, and no
differences were observed between groups (Table 2). Through the
exercise bout, the plasma FA oxidation was similar in the two groups
(Table 2), as was the proportion of FA oxidized. The estimated
oxidation of lipid sources other than plasma FA remained constant
through exercise and was not significantly different between groups
(Table 2).
Leg substrate balance.
The substrate oxidation across the leg through the exercise bout is
depicted in Fig. 3, and the muscle
glycogen contribution is based on an estimated active muscle mass of
4.6 kg. The muscle mass active during exercise was estimated from
plasma glucose uptake, the measured glycogen breakdown, and total
carbohydrate oxidation, determined by indirect calorimetry. The
substrate that is not covered in the sum of measured muscle substrates
amounts to 15 ± 6% in the Fat-Carbohydrate and 6 ± 13% in
Carbohydrate group.
View larger version (24K):
[in this window]
[in a new window]
|
Fig. 3.
Leg substrate utilization during 60 min bicycle exercise
in the fasted state after 8 wk of training and adaptation to Fat-CHO or
CHO diets. The contribution from glycogen was determined as net
breakdown multiplied by the estimated active muscle mass of the leg
(4.6 kg). Plasma FA contribution was calculated as the leg total FA
uptake multiplied by the whole body %FA oxidized assessed by stable
isotope tracer methodology, and contribution of blood glucose and
lactate was calculated from blood flow multiplied by the arteriovenous
difference through the exercise. The partition between nonprotein
fat-carbohydrate oxidation calculated from whole body respiratory
exchange ratio is indicated on the right side of each dietary
treatment. Values are means. * P < 0.05 Fat vs.
CHO.
|
|
| |
DISCUSSION |
In the present study, fuel utilization in the exercising leg was
studied in two groups after prolonged adaptation to either fat- or
carbohydrate-rich diet combined with training, followed by a
carbohydrate-rich diet for an additional week in both groups. The main
findings were that preexercise muscle glycogen stores were increased
and leg glucose uptake during submaximal exercise decreased in the
group that switched to a carbohydrate diet after the fat adaptation
(Fat-Carbohydrate) compared with the group that remained on the
carbohydrate-rich diet (Carbohydrate). The tracer-determined uptake of
plasma long chain FAs (LCFA) across the leg was not significantly
different between the groups and neither was whole body tracer
determined Rd of plasma LCFA for palmitate. The RER values
demonstrated that the overall proportions of carbohydrate and lipid
utilization were similar in the two groups during exercise.
The similar RER values during exercise in the two groups are in
agreement with our laboratory's previous findings (16), in which a switch to a carbohydrate-rich diet after prolonged fat diet
adaptation completely abolished the increased fat oxidation during
exercise that was observed after prolonged fat adaptation (17). Burke and colleagues (5) found that RER
values during 2 h cycling at 70% of peak oxygen uptake in
well-trained athletes were lower after short-term fat adaptation (5 days) followed by 1 day of carbohydrate loading (70-75% energy % carbohydrate) compared with 6 days of adaptation to the
carbohydrate-rich diet. In addition, they reported that
tracer-determined whole body glucose uptake during exercise was similar
in the two groups. Thus Burke and colleagues demonstrated that the
observed decrease in carbohydrate oxidation was solely due to muscle
glycogen sparing in the fat-adapted group. Intake of a fat-rich diet
followed by carbohydrate loading for 1 day also lowered carbohydrate
oxidation during a 4-h bicycle exercise trial at 65% of peak oxygen
uptake compared with when carbohydrates were ingested during the whole
period (6). In contrast, we found that muscle glycogen
utilization in absolute numbers was similar between groups, but blood
glucose uptake across the leg was lower during exercise in
Fat-Carbohydrate than in Carbohydrate. The latter finding is most
likely due to the increased muscle glycogen concentration in the
Fat-Carbohydrate group (Table 3) because studies have demonstrated that
glucose uptake during muscle contractions is inversely related to the
muscle glycogen level (11, 13). The molecular mechanism
behind the effect of glycogen on glucose uptake has been shown to
involve impaired GLUT-4 translocation to the surface membrane
(11). Increased muscle glycogen stores have also been
demonstrated to be associated with decreased activation of
5'-AMP-activated protein kinase during contractions (10,
25), but whether this is related to decreased muscle glucose
uptake during exercise is uncertain (10, 23, 25). The
difference in muscle glucose uptake between the two groups could not be
ascribed to differences in total muscle GLUT-4 protein content because
this was similar in the two groups (Table 3). However, it does not rule
out the possibility of either recruitment differences in GLUT-4
transporters or differential distribution of the pools of transporters
between the muscle membrane and intracellular stores. High plasma
concentrations of LCFA have been shown to decrease muscle glucose
uptake during exercise (14), but, because these were
similar in the two groups during exercise (Fig. 2), group differences
in leg glucose uptake in the present study could not be ascribed to
differences in plasma LCFA.
Despite the higher resting muscle glycogen concentration in the
Fat-Carbohydrate group, the actual muscle glycogen breakdown during
exercise was not significantly different between groups. It has been
demonstrated that muscle glycogen breakdown rate normally is related to
preexercise muscle glycogen concentrations both in vitro (18,
24) and in vivo (15). Thus the absence of increased
muscle glycogen breakdown in the Fat-Carbohydrate group suggests that
muscle glycogen breakdown may be attenuated after fat diet adaptation
followed by carbohydrate loading. This interpretation is supported by
our laboratory's previous study (16) in which exhaustion
in subjects on a similar diet as the present Fat-Carbohydrate group
occurred in spite of very high (~500 µmol/g dry wt) muscle glycogen
levels. It thus appears that prolonged adaptation to a fat-rich diet,
even when switching to a carbohydrate-rich diet for an additional week,
affects muscle metabolism during exercise in such a way that muscle
glycogen breakdown is impaired. The molecular mechanisms behind this
phenomenon remain to be established.
It might be argued that if glucose utilization is decreased and the
overall oxidation of carbohydrates is unchanged in the Fat-Carbohydrate
group compared with the Carbohydrate group, then glycogen breakdown
must be increased. This was not found. However, the decrease in leg
glucose uptake in terms of energy is rather small (Fig. 3), and it is
quite possible that a similarly small increase in muscle glycogenolysis
is missed. Furthermore, the overall combustion of carbohydrates and fat
is calculated from whole body RER values that may not be exactly the
same as respiratory quotient in the muscle and therefore small changes
in the balance of carbohydrate vs. fat combustion in the leg may not
necessarily be picked up at the level of pulmonary gas exchange.
However, the similar isotope-derived calculated leg and whole body
uptake of LCFA and absence of measurable breakdown of intramyocellular triacylglycerol in the both groups supports the concept of no significant difference in overall carbohydrate and fat utilization in
the two groups.
In our previous study (17), breakdown of VLDL-TG across
the leg during exercise could account for a significant part of the
lipid utilization across the leg after prolonged fat diet adaptation.
However, in the present study there was no measurable breakdown of
VLDL-TG across the leg in either group. Apparently, 1 wk of
carbohydrate-rich diet after the fat adaptation is enough to abolish
the significant contribution of VLDL-TG to energy provision during
exercise in the fat-adapted state. Intramyocellular triacylglycerol breakdown during exercise was not measurable in the present and several
previous studies performed in men (2, 19, 26, 30, 32).
When calculating the relative contribution of oxidized substrates in
the two groups, there is an apparent lack of substrates (Fig. 3). The
extent to which this may be accounted for by uptake of VLDL-TG or
breakdown of intramyocellular triacylglycerol, which were both too
small to be measurable, or by FAs liberated from adipocytes adherent to
muscle cells, as suggested by Kiens et al. (19), is not
possible to determine from the available data. In any case, the
contribution of these potential sources of energy substrates to
oxidation is likely to be minor. Finally, it should be considered that
because the pulmonary RER may not be exactly the same as leg
respiratory quotient, the balance between carbohydrate and lipid
combustion may not be entirely correct and therefore the apparent
missing substrate may not necessarily be lipid.
In conclusion, when switching to a carbohydrate diet for a week after
prolonged fat adaptation, overall fat and carbohydrate utilization
during submaximal exercise was not different compared with the group
that consumed the carbohydrate-rich diet all along. However, muscle
glycogen concentration was increased by 27% on average in the group
switching from fat adaptation to a carbohydrate-rich diet. This did not
lead to increased muscle glycogen utilization during exercise but to a
decrease in utilization of blood glucose. Thus it seems as if prolonged
fat adaptation leads to impaired muscle glycogen utilization, which is
not remedied by 1 wk of carbohydrate-rich diet.
| |
ACKNOWLEDGEMENTS |
The skilled technical assistance of Martin Rollo, Irene Beck
Nielsen, Betina Bolmgren, Inge Reich, Winnie Tågerup, and Nina Pluzek
is acknowledged.
| |
FOOTNOTES |
The study was supported by grants from the Danish National Research
Foundation Grant no. 504-14, the Danish Research Academy (J.nr.
77-7711), Team Danmark and the Danish Sports Research Council J.nr.
94-1-09, the UK Medical Research Council, and the Wellcome Trust.
Address for reprint requests and other correspondence:
J. W. Helge, Copenhagen Muscle Research Centre, H:S State
Hospital Section 7652, Blegdamsvej 9, DK-2100 Copenhagen Ø, Denmark
(E-mail: jhelge{at}mgi.ku.dk).
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.
August 2, 2002;10.1152/japplphysiol.00420.2002
Received 14 May 2002; accepted in final form 31 July 2002.
| |
REFERENCES |
1.
Andersen, P,
and
Saltin B.
Maximal perfusion of skeletal muscle in man.
J Physiol
366:
233-249,
1985.
2.
Bergman, BC,
Butterfield GE,
Wolfel EE,
Casazza GA,
Lopaschuk GD,
and
Brooks GA.
Evaluation of exercise and training on muscle lipid metabolism.
Am J Physiol Endocrinol Metab
276:
E106-E117,
1999.
3.
Bergström, J.
Muscle electrolytes in man: determined by neutron activation analysis on needle biopsy specimens. A study on normal subjects, kidney patients and patients with chronic diarrhea.
Scand J Clin Lab Invest Suppl
68:
11-13,
1962.
4.
Brooke, MH,
and
Kaiser KK.
Three "myosin ATPase" systems: the nature of their pH lability and sulfhydryl dependence.
J Histochem Cytochem
18:
670-672,
1970.
5.
Burke, LM,
Angus DJ,
Cox GR,
Cummings NK,
Febbraio MA,
Gawthorn K,
Hawley JA,
Minehan M,
Martin DT,
and
Hargreaves M.
Effect of fat adaptation and carbohydrate restoration on metabolism and performance during prolonged cycling.
J Appl Physiol
89:
2413-2421,
2000.
6.
Carey, AL,
Staudacher HM,
Cummings NK,
Stepto NK,
Nikolopoulos V,
Burke LM,
and
Hawley JA.
Effects of fat adaptation and carbohydrate restoration on prolonged endurance exercise.
J Appl Physiol
91:
115-122,
2001.
7.
Christensen, EH,
and
Hansen O.
Arbeitsfähigkeit und ernärung.
Skandinavishes Archiv für Physiolgie
81:
160-171,
1939.
8.
Christensen, NJ,
Vestergaard P,
Sorensen T,
and
Rafaelsen OJ.
Cerebrospinal fluid adrenaline and noradrenaline in depressed patients.
Acta Psychiatr Scand
61:
178-182,
1980.
9.
Coyle, EF,
Jeukendrup AE,
Wagenmakers AJM,
and
Saris WHM
Fatty acid oxidation is directly regulated by carbohydrate metabolism during exercise.
Am J Physiol
36:
E268-E275,
1997.
10.
Derave, W,
Ai H,
Ihlemann J,
Witters LA,
Kristiansen S,
Richter EA,
and
Ploug T.
Dissociation of AMP-activated protein kinase activation and glucose transport in contracting slow-twitch muscle.
Diabetes
49:
1281-1287,
2000.
11.
Derave, W,
Lund S,
Holman GD,
Wojtaszewski J,
Pedersen O,
and
Richter EA.
Contraction-stimulated muscle glucose transport and GLUT-4 surface content are dependent on glycogen content.
Am J Physiol Endocrinol Metab
277:
E1103-E1110,
1999.
11a.
Drabkin, DL,
and
Austin JH.
Spectrophotometric studies. II. Preparations from washed cells: nitric oxide, hemoglobin, and sulfhemoglobin.
J Biol Chem
112:
51-55,
1935.
12.
Frayn, K.
Calculation of substrate oxidation rates in vivo from gaseous exchange.
J Appl Physiol
55:
628-634,
1983.
13.
Gollnick, P,
Pernow B,
Essén B,
Jansson E,
and
Saltin B.
Availability of glycogen and plasma FFA for substrate utilization in leg muscle of man during exercise.
Clin Physiol
1:
27-42,
1981.
14.
Hargreaves, M,
Kiens B,
and
Richter EA.
Effect of increased plasma free fatty acid concentrations on muscle metabolism in exercising men.
J Appl Physiol
70:
194-201,
1991.
15.
Hargreaves, M,
McConell G,
and
Proietto J.
Influence of muscle glycogen on glycogenolysis and glucose uptake during exercise in humans.
J Appl Physiol
78:
288-292,
1995.
16.
Helge, JW,
Richter EA,
and
Kiens B.
Interaction of training and diet on metabolism and endurance during exercise in man.
J Physiol
292:
293-306,
1996.
17.
Helge, JW,
Watt PW,
Richter EA,
Rennie MJ,
and
Kiens B.
Fat utilization during exercise; adaptation to fat-rich diet increases utilization of plasma FA and VLDL-TG.
J Physiol
537:
1009-1020,
2001.
18.
Hespel, P,
and
Richter EA.
Glucose uptake and transport in contracting, perfused rat muscle with different precontraction glycogen concentrations.
J Physiol
427:
347-59: 347-359,
1990.
19.
Kiens, B,
Essen-Gustavsson B,
Christensen NJ,
and
Saltin B.
Skeletal muscle substrate utilization during submaximal exercise in man: effect of endurance training.
J Physiol
469:
459-478,
1993.
20.
Kiens, B,
and
Lithell H.
Lipoprotein metabolism influenced by training-induced changes in human skeletal muscle.
J Clin Invest
83:
558-564,
1989.
21.
Kiens, B,
and
Richter EA.
Types of carbohydrate in an ordinary diet affect insulin action and muscle substrates in humans.
Am J Clin Nutr
63:
47-53,
1996.
22.
Lowry, OH,
and
Passonneau JV.
A Flexible System of Enzymatic Analysis. New York: Academic, 1972.
23.
Richter, EA,
Derave W,
and
Wojtaszewski JF.
Glucose, exercise and insulin: emerging concepts.
J Physiol
535:
313-322,
2001.
24.
Richter, EA,
and
Galbo H.
High glycogen levels enhance glycogen breakdown in isolated contracting skeletal muscle.
J Appl Physiol
61:
827-831,
1986.
25.
Richter, EA,
McDonald C,
Kiens B,
Hardie G,
and
Wojtaszewski JFP
Dissociation of 5'AMP-activated protein kinase activity and glucose clearance in human skeletal muscle during exercise (Abstract).
Diabetes
50, Suppl 2:
A62,
2001.
26.
Roepstorff, C,
Steffensen CH,
Madsen M,
Stallknecht B,
Kanstrup IL,
Richter EA,
and
Kiens B.
Gender differences in substrate utilization during submaximal exercise in endurance-trained subjects.
Am J Physiol Endocrinol Metab
282:
E435-E447,
2002.
27.
Romijn, JA,
Coyle EF,
Sidossis LS,
Gastadelli A,
Horowitz JF,
Endert E,
and
Wolfe RR.
Regulation of endogenous fat and carbohydrate metabolism in relation to exercise intensity and duration.
Am J Physiol Endocrinol Metab
265:
E380-E391,
1993.
28.
Scrimgeour, CM,
and
Rennie MJ.
Automated measurement of the concentration and 13C enrichment of carbon dioxide in breath and blood samples using the Finnigan MAT Breath Gas Analysis System.
Biomed Environ Mass Spectrom
15:
365-367,
1988.
29.
Sidossis, LS,
Coggan AR,
Gastadelli A,
and
Wolfe RR.
A new correction factor for use in tracer estimations of plasma fatty acid oxidation.
Am J Physiol Endocrinol Metab
269:
E649-E655,
1995.
30.
Standl, E,
Lotz N,
Dexel T,
Janka HU,
and
Kolb HJ.
Muscle triglycerides in diabetic subjects: effects of insulin deficiency and exercise.
Diabetologia
18:
463-469,
1980.
31.
Steele, R.
Influences of glucose loading and of injected insulin on hepatic glucose output.
Ann NY Acad Sci
82:
420-432,
1959.
32.
Steffensen, CH,
Roepstorff C,
Madsen M,
and
Kiens B.
Myocellular triacylglycerol breakdown in females but not in males during exercise.
Am J Physiol Endocrinol Metab
282:
E634-E642,
2002.
33.
Wieland, O.
Glycerol assay.
In: Methods of Enzymatic Analysis, edited by Bergmeyer HV.. New York: Academic, 1974, p. 1404-1406.
34.
World Health Organization.
Energy and protein requirements: report of a joint FAO/WHO/UNU expert consultation.
Technical Report Series
724:
1-206,
1985.
J APPL PHYSIOL 93(5):1797-1805
8750-7587/02 $5.00
Copyright © 2002 the American Physiological Society
TOP
ABSTRACT
INTRODUCTION
![R<SUB>a</SUB> = <FR><NU>F − V [(C<SUB>palm</SUB> <IT>t</IT><SUB>2</SUB> + C<SUB>palm</SUB> <IT>t</IT><SUB>1</SUB>)/2] [(E<IT>t</IT><SUB>2</SUB> − E<IT>t</IT><SUB>1</SUB>) (<IT>t</IT><SUB>2</SUB><IT>−t</IT><SUB>1</SUB>)]</NU><DE>(E<SUB>2</SUB> + E<SUB>1</SUB>)/2</DE></FR>](/content/vol93/issue5/fulltext/1797/img001.gif)
![= <FR><NU>[(C<SUB>palm art</SUB> × E<SUB>palm art</SUB> − C<SUB>palm vein</SUB> × E<SUB>palm vein</SUB>]</NU><DE>C<SUB>palm art</SUB> × E<SUB>palm art</SUB></DE></FR>](/content/vol93/issue5/fulltext/1797/img008.gif)
