Fasting inhibits
and anaplerosis
insulin-mediated
glycolysis
in human skeletal muscle
CHARLES
E. CASTILLO,
ABRAM
KATZ,
MARK
K. SPENCER,
ZHEN YAN, AND BULANGU
L. NYOMBA
Clinical Diabetes and Nutrition
Section, National Institute of Diabetes, Digestive, and Kidney Diseases,
National Institutes of Health, Phoenix, Arizona 85106; and Department of Kinesiology,
University of Illinois, Urbana, Illinois 61801
CASTILLO,~HARLESE., ABRAM KATZ,MARK K. SPENCER, muscle (6, 24), as well as in the perfused rat hindlimb
ZHENYAN,ANDBULANGUL. NYOMBA.Fastinginhibitsinsulin(6). These latter studies showed that starvation resulted
mediated glycolysis and anaplerosis in human skeletal muscle.
in increases in insulin-mediated glycolysis and glycogenAm. J. Physiol. 261 (Endocrinol.
Metab. 24): E598-E605,
esis, demonstrating that both pathways for glucose uti1991.-Euglycemic
(~5.5 mM) hyperinsulinemic
(60 mu. mS2
lization had become hypersensitive to insulin (6, 24).
min-l) clamps were performed for 2 h after a 10-h fast and
Consistent with these results were the findings that, after
after a prolonged (72-h) fast. Biopsies were obtained from the
starvation, insulin binding (at submaximal concentraquadriceps femoris muscle before and after each clamp. The
tions of insulin) to rodent skeletal muscle was increased
rate of whole body glucose disposal was -50% lower during the
(6, 24), as was the insulin-mediated activation of 2clamp after the 72-h fast (P 5 0.001). The increase in carbohydrate (CHO) oxidation (which is proportional
to glycolysis)
deoxy-D-glucose transport (24). Similarly, starvation reduring the clamp after the 10-h fast (to 13.8 t 1.5 pmolkg
fat
sults in an increased expression of at least two distinct
free mass-l l rein-l) was completely abolished during the clamp
glucose transporters (adipose muscle and erythrocyte
after the 72-h fast (1.7 t 0.6; P 5 0.001). During the clamp
brain) in rat skeletal muscle (7). Insofar as starvation
after the 10-h fast, postphosphofructokinase
(PFK) intermeresults in insulin resistance in intact humans, the applidiates and malate in muscle increased, whereas glutamate decability of the results from rodent preparations to the
creased (P 5 0.05-0.001 vs. basal) and citrate did not change.
understanding of insulin-mediated carbohydrate (CHO)
During the clamp after the 72-h fast, there were no significant
changes in post-PFK intermediates
or glutamate (P > 0.05 vs. metabolism in humans is questionable.
The purpose of this study was to determine the effects
basal), but there was a decrease in citrate (P 5 0.01 vs. basal).
of short-term starvation on basal and insulin-mediated
Euglycemic hyperinsulinemia
increased glycogen synthase fractional activity in muscle under both conditions but to a greater
CHO metabolism in humans. Specifically, we were interextent after the 72-h fast (P 5 0.01). It is concluded that insulin
ested in determining whether the different pathways of
(after 10-h fast) increases glycolytic flux and the content of glucose utilization
(glycogenesis and CHO oxidation)
malate in muscle, which is probably due to increased anaplewere affected in a similar manner by starvation. Measrosis. The decrease in citrate during hyperinsulinemia
after the
urements of a number of metabolites and enzyme activi72-h fast indicates that insulin activated the tricarboxylic
acid
ties in muscle were used to identify potential regulatory
(TCA) cycle but that the replenishment
of TCA cycle intersites in the starvation-mediated effects.
mediates normally observed (10-h fast) was blocked, possibly
l
due to inadequate provision of three-carbon
intermediates
for
anaplerosis. These data demonstrate that prolonged fasting can
increase and decrease insulin-mediated
activation of processes
associated with CHO metabolism in human skeletal muscle.
lactate;
dation
malate;
citrate;
glycogen
synthase;
carbohydrate
oxi-
IT IS WELL DOCUMENTED that in intact humans shortterm starvation results in a decrease in whole body basal
and insulin-mediated rates of glucose disposal (5). After
starvation, basal and insulin-mediated glucose uptake by
intact human skeletal muscle is reduced (28, 29, 37).
Studies on rodents, using either the perfused rat hindlimb or isolated skeletal muscle in vitro, have demonstrated that glucose utilization in the absence or presence
of insulin is not impaired after starvation (2, 11). In fact,
at submaximal concentrations of insulin, glucose utilization is increased both in isolated mouse and rat soleus
E598
0193~1849/91
$1.50
Copyright
METHODS
Subjects. Eight men, whose mean (range) age, height,
weight, percent body fat, and plasma glucose concentration 2 h after ingesting a solution containing 75 g of
glucose were, respectively, 29 yr (22-40), 174 cm (169180), 71.7 kg (63.1-80.3), 15.7% (8.2-20.6), and 5.8 mmol/
1 (4.7-6.9), participated in the study after giving voluntary consent. The subjects’ health was assessedby physical examination and routine hematologic, electrocardiograph, and urine tests. The experimental protocol was
approved by the ethics committee of the National Institutes of Health.
Experimental design. All subjects were studied on two
occasions during their stay on the metabolic ward (-7
days). The first study was preceded by at least 3 days on
a weight-maintenance diet (45% CHO, 40% fat, 15%
protein). After an overnight fast (-10 h), a euglycemic
hyperinsulinemic clamp was performed. Briefly, at 0600
0 1991 the American
Physiological
Society
FASTING
AND
CARBOHYDRATE
h and after the subject had voided, a catheter was placed
in an antecubital vein for infusion of insulin, glucose,
and [3-3H]glucose.
Another catheter was placed retrograde in a dorsal hand vein of the contralateral
hand for
blood sampling. To arterialize the blood, the hand was
kept in a warming
box at 70°C. A primed (30 &i),
continuous
infusion of [3-3H]glucose
(0.3 &i/min)
was
started at -120 min and was maintained
for 240 min
(i.e., until 120 min). At 0 min, a primed (958 mu/m’)
continuous
infusion of insulin (60 mU m-’ min-‘) was
started and maintained
for 120 min. A variable 20%
glucose infusion was performed between 0 and 120 min
to achieve an arterialized plasma glucose concentration
of -5.5 mmol/l. Samples for plasma glucose were obtained every 5 min throughout
the insulin infusion.
Samples for determination
of plasma [ 3-3H] glucose
specific activity were drawn every 10 min from -40 to 0
min and from 80 to 120 min. Rates of glucose infusion
and appearance and CHO and fat oxidation
(indirect
calorimetry)
were estimated at the same time. Glucose
disposal (i.e., disappearance of glucose from plasma) was
estimated from the specific activity of [3-3H]glucose
in
plasma and its rate of infusion using Steele’s non-steadystate equations
(34), assuming
a glucose distribution
volume of 100 ml/kg. The total rate of glucose appearance was never higher than the rate of glucose infusion
in any subject after the 10-h fast; therefore the rate of
glucose infusion is used to reflect glucose disposal. After
the 72-h fast (see below), however, the rate of glucose
appearance was slightly higher than the glucose infusion
rate in four subjects (suggesting incomplete inhibition of
endogenous glucose production).
In these four subjects
the rate of glucose appearance is used to reflect glucose
disposal, whereas in the other four subjects the rate of
glucose infusion is used to reflect glucose disposal. Further details on the clamp procedure, determination
of
body composition
(underwater
weighing),
methods for
analysis of [ 3-3H] glucose specific activity, indirect calorimetry
measurements,
and calculations
are provided
elsewhere (25).
Biopsies from the lateral aspect of the quadriceps
femoris muscle were obtained with the needle biopsy
technique. Briefly, after local anesthesia
(lidocaine 10
mg/ml), incisions at biopsy sites on both thighs were
made, and biopsies were obtained before (0 min) and at
the end of the clamp. After the clamp the subjects were
allowed to eat until nightfall. The next morning they ate
breakfast;
thereafter,
the subjects started a 72-h fast.
They were allowed to drink all fluids that did not contain
any calories. After 72 h, at the same time of day (i.e.,
0600 h), the same clamp procedure, as described above,
was repeated. Thus the clamps were not randomized.
This is because we did not know how long it would take
to completely recover from the 72-h fast. Therefore the
10-h fast experiments
were always performed first. It is
not likely that lack of randomization
significantly
affected the results, because, had the subjects not fasted
for 72 h after the initial clamp (i.e., if they would have
consumed the controlled diet described above), virtually
all of the measured values measured in the basal state
72 h after the initial clamp would have been the same as
l
l
METABOLISM
IN
MUSCLE
E599
those obtained before the clamp after the 10-h fast (31,
33)
dnalytical
methods. All biopsies were frozen in liquid
N2 and stored at -70°C until analysis. The samples were
freeze-dried, dissected free from nonmuscle constituents
(blood, fat, connective tissue), and powdered. The powder
was thoroughly mixed and, when sufficient material was
available, was divided into six aliquots. One aliquot was
extracted with 0.5 M perchloric acid and was neutralized
with KHC03. The neutralized extract was used for fluorometric enzymatic analyses of metabolites
(3, 26). The
second aliquot was digested in 50 mM NaOH (SOOC) for
20 min. The extract was neutralized with acetic acid and
was assayed spectrophotometrically
for fructose 2,6-diphosphate
(F-2,6-&)
using pyrophosphate-dependent
phosphofructokinase
(PFK) from potato tubers (35). The
third aliquot was digested in 1 M KOH (6O”C), neutralized with HCl, and used for enzymatic analysis of total
carnitine
(spectrophotometrically)
(3). In another part
of this neutralized extract, glycogen was hydrolyzed enzymatically
and was assayed enzymatically
for glucose
(13). Glycogen was expressed as millimoles glucosyl units
per kilogram dry weight. The fourth aliquot was used for
measurement
of glycogen synthase (GS) activity using
filter paper (see Ref. 22). Briefly, powder was homogenized with a buffer containing 30% (vol/vol) glycerol, 10
mM EDTA, and 50 mM KF, pH 7, at 4°C. The homogenate was centrifuged at 10,000 g for 20 min at 4°C. The
supernatant
was diluted with a buffer containing
50
mM tris(hydroxymethyl)aminomethane
(Tris), 20 mM
EDTA, and 130 mM KF, pH 7.8, at 4°C and was then
used for assay of GS. Enzyme (25 ,ul) was added to 50 ~1
of a reaction buffer that contained a low glucose 6phosphate (G-6-P) concentration
(0.17 mM; GSI,,) and
a high G-6-P concentration
(7.2 mM; GShigh). UDPglucose concentration
in the buffer was 0.20 mM. Enzyme activity was estimated from the incorporation
of
UDP-[14C]glucose
into glycogen at 30°C. The fractional
activity (GSF) is GSlow/GShigh.’
The fifth aliquot was used for analysis of GS phosphatase (GSP), which was assayed as previously
described
(see Ref. 22). Commercial
rabbit GS-D (i.e., dependent
on G-6-P; Sigma G2259) was purified as described elsewhere (22) and was -95% G-6-P dependent when assayed in the presence of 7.0 mM UDP-glucose
with and
without 7.2 mM G-6-P. Muscle powder was homogenized
with a ground-glass
homogenizer in a buffer (133 pl/mg
dry wt) containing 50 mM Tris, 10 mM EDTA, and 50
mM 2-mercaptoethanol,
pH 7.8, at 4°C. The homogenate
was centrifuged
at 10,000 g for 20 min at 4°C. Purified
GS-D (25 mu, 8 ~1) were preincubated
with 0.3% glycogen, 10 mM Tris, 1 mM EDTA, and 5 mM dithiothreitol,
pH 7.8 (total volume 75 ~1) for 5 min at 30°C. The GSP
reaction was then started by transferring
100 ~1 of extract
supernatant
into the preincubated
reaction mixture. The
reaction was stopped right after the start of reaction and
after 15 min incubation by diluting 50 ,ul of the incubation mixture with 2,000 ,ul of 130 mM KF, 50 mM Tris,
and 20 mM EDTA, pH 7.8, at 4°C. Twenty-five
microliters were then used to determine GSP activity using
the GS assay (i.e., the change in GSI,,). Values are
expressed as micromoles
of glucose from UDP- [ 14C]glu-
E600
FASTING
AND
CARBOHYDRATE
case incorporated
into glycogen per kilogram dry weight
per minute squared.
The last aliquot was used for analysis of adenosine
3’,5’-cyclic
monophosphate
(CAMP)-dependent
protein
kinase (CAMP-PK),
which was assayed using kemptide
as substrate (see Ref. 24). Muscle powder was homogenized in a buffer (60 pl/mg dry wt) containing
10 mM
Tris, 10 mM EDTA, 100 mM KF, and 1% charcoal, pH
7.6, using a ground-glass
homogenizer. The homogenate
was centrifuged at 12,800 g for 10 min at 4°C. Extract
supernatant
(15 ~1) were incubated with 85 ~1 of reaction
mixture, giving a final concentration
of 200 PM kemptide
(Sigma, K1127), 0.3 mM [32P]ATP (sp act -20 mCi/
mmol; Amersham),
50 mM Z-(N-morpholino)ethanesulfonic acid, and 8 mM MgC12, pH 6.5, in the absence
or presence of 0.2 and 100 PM CAMP at 30°C for 5 min.
Preliminary
data showed that CAMP-PK
was saturated
at 100 PM CAMP (data not shown). To stop the reaction,
60 ~1 of the incubation mixture were applied to a disk of
Whatman-P81
paper. The disks were washed with 150
mM phosphoric acid, rinsed with 95% ethanol, and dried.
The radioactivity
was determined in a Packard scintillation counter. CAMP-PK
activity was expressed as micromoles 32P incorporated
into kemptide per kilogram
dry weight per minute. All enzyme activities were linear
with time and extract volume (data not shown).
The
interassay coefficient of variation for muscle metabolites
and enzymes is generally <lo% (see Ref. 13 and Katz,
unpublished observations).
All metabolite contents (except for pyruvate, lactate,
and glucose, which are also present in the extracellular
compartment)
were adjusted to the mean total creatine
content (sum of phosphocreatine
and creatine) for the
whole material [ 125.7 t 1.8 (SE) mmol/kg dry wt]. This
adjustment
was performed to correct for variability
in
blood, connective tissue, or other solid nonmuscle constituents between biopsies.
Plasma glucose was determined with the glucose oxidase method using a Beckman glucose analyzer (Beckman Instruments,
Fullerton,
CA), and plasma glucose
was determined by radioimmunoassay
using a Concept 4
automated
analyzer (Micromedic
Systems,
Norsham,
PA). Plasma free fatty acids were determined enzymatitally (see Ref. 19).
Statistics. Significant differences
(P IS 0.05) were determined with the paired t test. Values are presented as
means t SE unless otherwise indicated.
RESULTS
The 72-h fast resulted in a significant decrease in body
weight (from 71.7 t 1.9 to 68.5 t 1.9 kg; P 5 0.001). No
significant difference in urinary nitrogen output was
detected as a result of fasting [control, 7.2 t 0.6 mg*kg
fat-free mass (FFM)-lomin-l;
fasting, 8.3 t 0.7; P >
0.051. Percent body fat was only determined before the
first clamp, and it was not deemed valid to use this value
to estimate FFM after the 72-h fast (this would have
resulted in an overestimate in the loss of lean body mass).
To determine the loss of FFM during the 3-day fast, the
urinary nitrogen excretion was corrected for insensible
nitrogen loss, and total protein loss was estimated using
METABOLISM
IN
MUSCLE
a value of 6.25 g protein/g nitrogen. FFM after the 3-day
fast was then calculated by subtracting protein loss from
the initial FFM. FFM was estimated to decrease from
60.4 t 1.9 kg before the 3-day fast to 59.3 t 1.8 kg after
the fast (P 5 0.001).
The 72-h fast resulted in a significant decrease in basal
endogenous glucose production and insulin-mediated
glucose disposal (Table 1). During the clamp after the
10-h fast, endogenous glucose production was completely
suppressed, and there was no significant difference between treatments during the clamp. These data support
the previous conclusion that fasting-mediated insulin
resistance is relegated to peripheral tissues (5). The 72h fast resulted in a marked decrease in basal CHO
oxidation and completely blocked the increase in CHO
oxidation during the clamp. The 72-h fast did not affect
the basal rate of nonoxidative glucose disposal, and although nonoxidative glucose disposal during the clamp
was lower after the 72-h fast in six of the eight subjects,
the relative effect of the 72-h fast was not nearly as
dramatic as that on CHO oxidation. Similarly, the 72-h
fast resulted in increased rates of fat oxidation in the
basal state and completely abolished the insulin-mediated decrease in fat oxidation.
The effects of short-term starvation on the concentrations of metabolites and insulin in plasma in the basal
state (Table 2) are as previously described under similar
1. Effects of 72-h fast and insulin
on whole body metabolic rates
TABLE
Endogenous
glucose
production,
pmol . kg
FFM-l.
min-l
10 h
72 h
Glucose disposal, pmol kg
FFM-1
min-l
10 h
72 h
CHO 0X, pmol . kg FFM-1 a
min-’
10 h
72 h
CHO nonox, pmol . kg FFM-l.
min-l
10 h
72 h
Fat 0X, ymol . kg FFM-1
min-’
10 h
72 h
RER (VcoJVo2)
10 h
72 h
vog, ml kg FFM-l.
min-’
10 h
72 h
EE, Cal. kg FFM-l.
min-’
10 h
72 h
Basal
Clamp
16.3t0.4
11.6t0.4"
O.l+O.lf
1.7_t0.7f
l
l
16.3t0.4
11.6t0.4"
8.4t0.7
2.320.4"
13.8t1.5f
1.7t0.6"
7.9t0.7
9.320.3
25.3t3.2"
18.1t1.4f~a
2.9t0.4
4.5kO.3"
1.7kO.4"
4.720.3"
0.835t0.009
0.756t0.005"
0.899t0.017"
0.748t0.006"
3.18t0.10
3.35+0.0gb
3.33t0.09
3.36kO.10
15.2t0.5
15.7t0.4
16.1+0.5d
15.7kO.4
l
l
Values are means t SE for 8 individuals.
FFM, fat-free
mass. Fat,,,
fat oxidation,
assuming
palmitate
is being oxidized
(mol wt = 258).
CHO,,,,
carbohydrate
oxidation;
CHO,,,,,
nonoxidative
carbohydrate
disposal;
ko2,
CO2 production;
%702, volume
of 02 consumed;
RER,
respiratory
exchange
ratio; EE, energy expenditure.
a P I 0.05, b P s
0.01, 'P IS 0.001, vs. 10 h. d P 5 0.05, e P 5 0.01, f P 5 0.001, vs.
respective
basal value.
FASTING
AND
CARBOHYDRATE
METABOLISM
2. Effects of 72-h fast and insulin
on plasma metabolites
Basal
Values
are means
Clamp
5.3kO.l
3.9t0.2”
Basal
5.4t0.1
5.5t0.1f
0.25t0.02
0.87t0.06"
0.05+0.01f
0. 10+0.02a~f
0.71-r-0.08
1.02-c-0.06”
0.70t0.04
0.76+0.07b
5tl
2tlb
76t4f
72k4f
& SE for 8 individuals.
FFA,
free fatty
acids.
aP
5 0.05, bP 5 0.01, "P 5 0.001, vs. 10 h. eP 5 0.01, f P 5 0.001, vs.
respective
basal
value.
3. Effects of 72-h fast and insulin
on high-energy phosphates in muscle
TABLE
PCr
10 h
72 h
Cr
10 h
72 h
PCr/Cr
10 h
72 h
ATP
10 h
72 h
Basal
Clamp
80.2t2.1
78.2tl.4
78.6tl.5
79.1t2.0
45.51t2.1
47.5kl.4
47.1tl.4
1.80t0.11
1.69t0.08
1.6620.08
1.7320.12
24.2t0.6
24.320.5
23.9t0.7
24.1t0.8
Values are means t SE for 8 individuals
dry wt. PCr, phosphocreatine;
Cr, creatine.
E601
MUSCLE
TABLE 4. Effects of 72-h fast and insulin on
glycogenolytic and glycolytic intermediates in muscle
TABLE
Glucose, mmol/l
10 h
72 h
FFA, mmol/l
10 h
72 h
Lactate,
mmol/l
10 h
72 h
Insulin,
&J/ml
10 h
72 h
IN
46.6t2.0
and are given
in mmol/kg
Glycogen
10 h
72 h
Glucose
10 h
72 h
UDP-Glucose
10 h
72 h
Glucose l-phosphate
10 h
72 h
Glucose 6-phosphate
10 h
72 h
Fructose
6-phosphate
10 h
72 h
Fructose
1,6-diphosphate
10 h
72 h
DHAP
10 h
72 h
Glucose 1,6-diphosphate
10 h
72 h
Fructose
2,6-diphosphate
10 h
72 h
Values are means t SE for
kg dry wt, except for glucose
phate, which are in pmol/kg
phate. b P 5 0.01, ’ P s 0.001,
value.
Clamp
379tl6
328k15b
390tl7
322tl2"
2.520.2
2.3t0.2
2.420.4
3.0+0.3d
0.86t0.12
l.llt0.16
1.03-eo.10
0.96t0.11
1.5t0.3
1.7-co.3
2.2t0.2
1.8-eO.3
0.63t0.08
0.64t0.09
0.74t0.08
0.62t0.09
0.27t0.05
0.33kO.06
0.28t0.05
0.27t0.05
0.17t0.03
0.21t0.03
0.2220.03
0.18t0.03
83_t4
96k9
94&6
86t9
11.4kl.3
15.4+l.6b
11.8t0.9
12.4tl.O
6-8 individuals
and are given in mmol/
1,6-diphosphate
and fructose
2,6-diphosdry wt. DHAP,
dihydroxyacetone
phosvs. 10 h. d P i 0.05 vs. respective
basal
5. Effects of 72-h fast and insulin
on carnitines and free amino acids in muscle
TABLE
conditions (5). Starvation completely inhibited the insulin-mediated increase in plasma lactate.
Neither starvation nor insulin affected the contents of
high-energy phosphates in muscle (Table 3). Starvation
resulted in an -15% decrease in the basal content of
muscle glycogen (Table 4) and a significant increase in
F-2,6& but did not affect any of the other sugar phosphates. There were no significant changes in any of the
sugar phosphates or glycogen during either clamp. It
could be calculated, however, that the increase in muscle
glycogen during the clamp after the 10-h fast would
amount to only -5% of the basal value. Starvation
resulted in a small but significant decrease in free carnitine and a threefold increase in acetylcarnitine, as well
as decreasesin glutamate and aspartate in the basal state
(Table 5). After the 10-h fast, hyperinsulinemia resulted
in a significant decrease in glutamate. There was a trend
for acetylcarnitine to decrease during the clamp after
starvation, but the decrease was not significant.
Starvation did not affect any of the measured threecarbon glycolytic intermediates (including alanine) in
muscle in the basal state (Fig. 1). After the 10-h fast,
hyperinsulinemia resulted in significant increases in pyruvate, lactate, and alanine. On the other hand, starvation abolished the insulin-mediated increases in the
three-carbon intermediates. Starvation did not affect the
tricarboxylic acid (TCA) cycle intermediates in muscle
Basal
Total carnitine
10 h
72 h
Free carnitine
10 h
72 h
Acetylcarnitine
10 h
72 h
Glutamate
10 h
72 h
Aspartate
10 h
72 h
Values are means
dry wt. a P 5 0.05, ’
value.
.
Clamp
19.0tl.l
18.521.2
18.6kl.5
18.8kl.4
18.3tl.2
16.521.2”
17.6kl.4
16.6tl.3
0.21t0.05
0.69t0.11”
0.53t0.10”
11.5tl.O
0.22t0.04
7.9kO.9”
8.7t0.gf
7.2k0.8
1.7-r-0.1
0.9tO.l”
0.7kO.l"
t SE for 8 individuals
1.5kO.l
and are given
in mmol/kg
basal
P 5 0.001, vs. 10 h. f P 5 0.001 vs. respective
in the basal state (Fig. 2). After the 10-h fast, insulin
infusion did not affect the muscle citrate content, which
is consistent with previous observations (18, 33), but did
result in a significant increase in malate. However, after
starvation, insulin infusion resulted in a significant decrease in citrate. There was also a trend for malate to
decrease, but the decrease did not reach statistical significance.
E602
FASTING
O-4
AND
CARBOHYDRATE
METABOLISM
0.50
Pyruvate
IN
MUSCLE
Citrate
-NS-
-*1
0.25
-NS10
Lactate
f---*l-
--S-
Malate
0.25
8
Alanine
-NS-
-*1
I
I
0.00
Basal
Clamp
Basal
Control
Basal
Clamp
Control
Basal
Clamp
Fasted
FIG. 2. Effects
of 72-h fast and insulin
on citrate
and malate
in
muscle. Values are means t SE for 8 individuals.
Control,
after 10-h
fast; fasted, after 72-h fast; NS, not significant.
None of basal values
are significantly
different
between
treatments.
* P d 0.05; ** P 5 0.01.
1. Effects
of 72-h fast and insulin
on glycolytic
intermediates
in muscle. Values are means t SE for 6-8 individuals.
Control,
after
10-h fast; fasted, after 72-h fast; NS, not significant.
None of basal
values are significantly
different
between treatments.
* P 5 0.05; ** P
FIG.
Clamp
Fasted
TABLE 6. Effects of 72-h fast and insulin
on glycogen synthase activity in muscle
5 0.01.
Basal
Starvation resulted in a significant decrease in GSF in
the basal state, as well as a slight but significant decrease
in GShigh (Table 6). I nsulin activated GS under both
conditions. However, the insulin-mediated increase in
@SF after starvation was significantly greater than that
after the 10-h fast (A0.25 t 0.03 vs. A0.17 t 0.03; P 5
10 h
72 h
Gf%igh
10 h
72 h
GSlow/GShigh
10 h
72 h
0.01).
The activity of CAMP-PK (measured at 100 PM
CAMP) was statistically lower in the basal state after the
72-h fast (n = 4; Table 7). However, the difference was
small and is not considered to be of physiological significance. There was no indication of an altered activation
of the enzyme under any condition (O/100 and 0.2/100
CAMP). Complete data on GSP were available for only
three subjects; therefore significance testing was not
performed. The activity of GSP was lower after the 72h vs. the 10-h fast in the basal state for all three subjects.
DISCUSSION
Glucose disposal. This study confirms that starvation
results in a decrease in basal hepatic glucose production
(5). We now also demonstrate that fasting results in a
marked decrease in whole body CHO oxidation and an
increase in fat oxidation, such that caloric expenditure
1.4tO.l
0.870.2'
2.2+0.3d
2.1t0.2f
5.5t0.2
4.8+0.3b
5.0t0.3
5.1k0.2
0.25t0.02
0.42t0.04f
0.42+0.04f
0.16+0.02b
Values are means t SE for 8 individuals
and
dry wt-l min-l at 30°C. GS, glycogen synthase.
a glucose 6-phosphate
concentration
of 0.17 mM
b P 5 0.01, ’ P 5 0.001, vs. 10 h. d P 5 0.05, f P
basal value.
l
Clamp
are given in mmol kg
GSl,, was measured
at
and GShigh at 7.2 mM.
5 0.001, vs. respective
is not affected. Starvation did not affect the basal rate
of nonoxidative glucose disposal. However, it is not clear
what this component represents (glycogenesis, glycolysis,
or the pentose shunt pathway), or whether there were
any changes in the flux through these different pathways
within or between different organs.
The decrease in muscle glucose uptake in the basal
state after starvation (28, 29) is probably due to a decrease in substrate availability, as well as to a decrease
in plasma insulin, and possibly to changes in other
hormones (e.g., increase in growth hormone) (37). Based
on experiments in rats, there is no evidence to suggest
FASTING
AND
CARBOHYDRATE
METABOLISM
TABLE 7. Effects of 72-h fast and insulin on CAMP
protein kinase and GSP activities in muscle
Basal
CAMP-PK
10 h
72 h
CAMP-PK
10 h
72 h
CAMP-PK
10 h
72 h
GSP
10 h
72 h
Clamp
(O/100 PM)
(0.2/100
0.21t0.02
0.22t0.02
0.23zkO.01
0.21t0.01
0.62t0.04
0.67t0.03
0.64t0.01
0.71t0.04
pM)
(100 /JM)
97.1t12.3
102.2t12.6
95.4t13.8"
96.2t14.0
4641k21
332-c-46
347t19
278t32
Values are means & SE for 3-4 individuals
and are given in ,umol.
kg dry wt-l. min-’ [CAMP protein
kinase (CAMP-PK),
measured
at 0,
0.2, and 100 pM CAMP]
or pmol . kg dry wt-‘. min-’ [glycogen
synthase
phosphatase
(GSP)]
at 30°C. a P 5 0.05 vs. 10 h.
that fasting affects the capacity of the glucose transport
system, as judged by the increased expression of glucose
transporters (7) and rates of 2-deoxy-D-glucose uptake
(6,24). The increase in FFA concentration and oxidation
may also be contributing to the decrease in muscle glucose uptake (30). If this is correct, the decrease in glucose
uptake still cannot be explained by the classic glucosefatty acid cycle (30), because neither muscle citrate nor
G-6-P were increased in the basal state after the 72-h
fast.
This study confirms that starvation in humans results
in a marked decrease in insulin-mediated glucose disposal. During euglycemic hyperinsulinemia -90% of the
infused glucose is taken up by skeletal muscle (9). It
follows therefore that the decrease in insulin-mediated
glucose disposal after starvation is due primarily to a
lower glucose uptake by skeletal muscle.
Glycolysis. The effect of starvation on muscle glycolysis
can be evaluated as follows. If it is assumed that glycogen
decreased linearly over 72 h of fasting, then the glycogenolytic rate is estimated to be 0.01 mmol. kg dry wt-’
min.
This value is in agreement with the idea that
muscle glycogenolysis is negligible in the resting state
(8). The basal rate of glucose uptake by muscle has been
determined to be between 0.02 and 0.08 mmol. kg dry
wt-l . min-’ (1, 17). If it is assumed that the amount of
glucose going to glycogen is negligible (and this assumption becomes especially reasonable during the 3-day fast
because the activity of GS decreased), then glucose uptake can be estimated to account for 67-89% of glycolytic
flux in the basal state. Therefore, the finding that a 3day fast decreasesbasal muscle glucose uptake to a value
that is -35% of the control value (overnight fast) (28,
29), indicates that muscle glycolysis was lower after
starvation. Similarly, the findings that the rates of lactate and pyruvate release from the forearm are not altered during starvation, whereas the rate of CHO oxidation is decreased (28, 29), are also consistent with a
decreased glycolytic flux after starvation. The decrease
in muscle glycolysis may be due to the decrease in plasma
insulin, resulting in a decreased activation of PFK, a
regulatory enzyme for glycolysis. In this context, the
increase in F-2,6-P2 after starvation is puzzling, since
l
IN
MUSCLE
E603
this should, assuming all other factors are constant,
activate PFK (35).
After the 10-h fast, insulin infusion resulted in an
increase in whole body CHO oxidation as well as increases in the muscle contents of post-PFK intermediates, which indicate that muscle glycolysis was accelerated (i.e., PFK was activated). In a recent study, in which
insulin was infused at a lower rate (40 mUa rnm2.min-‘),
we could not detect significant increases in post-PFK
intermediates in muscle (33). However, at higher insulin
infusion rates (60 mu. rnm2.min-l), the increase in pyruvate and lactate become readily detectable (present
data). In contrast to the situation during prolonged insulin infusion (16), the activation of PFK during euglycemic hyperinsulinemia after the overnight fast in the
present study could not be attributed to increases in the
steady-state level of glucose 1,6-diphosphate, nor could
the activation of PFK be associated with increases in
any of the other measured activators of PFK [fructose
mono- and diphosphates and free ADP and AMP (based
on the phosphocreatine-to-creatine
ratio; see Ref. 36)]
or decreases in the inhibitors of PFK (citrate and ATP).
Either the changes in these metabolites necessary to
activate PFK are too small to be detected with our
techniques or insulin activates PFK by some other mechanism(s).
One of the dramatic effects of starvation was what
appeared to be a complete inhibition of insulin-mediated
glycolysis (no change in 3-carbon post-PFK intermediates or CHO oxidation). Because the concentrations of
fructose 6-phosphate and ATP were similar between
treatments during the clamp, the low glycolytic rate after
the 72-h fast is probably due to inadequate insulinmediated activation of PFK.
CHO oxidation. Starvation resulted in a significant
decrease in the basal rate of CHO oxidation. One possible
explanation for this is that the increased reliance on fat
oxidation resulted in an increase in acetyl-CoA, judged
from the increase in acetylcarnitine (2, 11). This should
result in an activation of pyruvate dehydrogenase kinase
and therefore an inhibition of pyruvate dehydrogenase
(14). In addition, a decrease in the availability of postPFK three-carbon intermediates (i.e., pyruvate), due to
a low glycolytic flux may also have contributed to the
low rate of CHO oxidation.
After the 10-h fast, insulin infusion did not alter the
content of citrate in muscle. However, there was a significant increase in malate. The increase in malate may
be due to an increased activation of anaplerotic reactions
(i.e., increased formation of TCA cycle intermediates),
which are dependent on the availability of three-carbon
intermediates such as pyruvate. The increases in pyruvate and alanine, coupled with the decrease in glutamate
(during the clamp after the 10-h fast) are consistent with
an increased flux through the alanine aminotransferase
reaction, resulting in the production of 2oxoglutarate.
2Oxoglutarate can then be used to increase malate and
other TCA cycle intermediates (32,33). It is also possible
that increased flux through the pyruvate carboxylase
reaction could have contributed to the increase in malate
and could have maintained levels of citrate. The activity
of pyruvate carboxylase is probably quite low in human
E604
FASTING
AND
CARBOHYDRATE
skeletal muscle, but because the postclamp biopsy was
taken 2 h after the basal biopsy, flux through pyruvate
carboxylase,
or other anaplerotic
reactions,
may have
been sufficient to contribute to the increase in malate.
The finding that citrate decreased during the clamp
after starvation indicates that insulin activated the TCA
cycle. It has been previously
documented
that insulin
activates the TCA cycle and respiration
in mitochondria
from diaphragm and heart muscle, as well as liver (4, 15,
27). [We did not measure a significant increase in whole
body oxygen consumption.
However,
a major increase,
even in the affected tissue, is not expected (4).]
The decrease in citrate during the clamp after starvation may be consequential
to lack of insulin-mediated
anaplerosis
or decreased
availability
of acetyl-CoA
within the mitochondria.
In either case, this could be
attributed to inadequate availability
of three-carbon
intermediates. Consistent
with this explanation are recent
findings on two patients with PFK deficiency in skeletal
muscle. During euglycemic hyperinsulinemia
(400 mu.
me2 .min-‘) there were decreases in acetylcarnitine
and
citrate in muscle. There were no increases in muscle
pyruvate
or lactate (20). We have recently described
other conditions
(exercise
and epinephrine
infusion)
where apparently increased glycolytic flux is required for
expansion of TCA cycle intermediates
(32,33). Thus the
present data suggest that starvation
attenuates insulinmediated stimulation
of CHO oxidation and anaplerosis
in muscle.
Glycogen synthase. Starvation
resulted in a small but
significant
decrease in the activity of GShigh. This has
also been observed in the soleus muscle of mouse after
48 h of starvation (24) and may be related to an increase
in muscle protein catabolism
(12). Starvation also resulted in a significant decrease in GSF in the basal state.
The mechanism for this is not clear. It may be due to
changes in the activities of the phosphatases
and kinases
that control the phosphorylation
state of GS and/or
changes in the substrate suitability
(i.e., affinity) of GS
for its phosphotransferases.
Indeed it has been shown
that 24 h of fasting results in a decreased activity of GS
phosphatase
and probably a decrease in the substrate
suitability
of endogenous GS for GSP in rat heart (10).
The present data, although limited, support the idea that
a decrease in GSP specific activity can at least partly
explain the decrease in GSF in the basal state after the
72-h fast. Moreover, the data also indicate that this
decrease in GSF is not due to an increased activation
state of CAMP-PK.
It might be argued that the decrease
in GSF is solely a consequence of the small decrease in
plasma insulin. We recently showed that, when glucose
infusion is superimposed
on somatostatin
infusion (increasing plasma glucose from -4 to 26 mM), plasma
insulin rises from 1 to 7 pU/ml (i.e., the increase is
similar to the decrease observed after starvation),
but
there is no significant change in GSF (19). These findings
suggest that the decrease in GSF is not due simply to
acute effects by small changes in insulin.
Interestingly,
GSF and GSI,, increased to the same
values in the two clamps and to larger extents during the
clamp after the 72-h fast. These data indicate that the
activation of muscle GS by insulin is not a consequence
METABOLISM
IN
MUSCLE
of increases in glucose uptake and/or utilization
(24).
We could not associate the increase in GSF during the
clamp with increases in GSP or decreases in CAMP-PK
activity. It should be noted that the clamp biopsies were
taken 2 h after onset of insulin infusion, and thus the
presumably early changes in the kinase and phosphatase
activities were not detected (21, 23).
In conclusion, these data demonstrate
that starvation
causes bidirectional
changes in insulin-mediated
activation of enzymes and metabolic pathways associated with
CHO metabolism in human skeletal muscle. Specifically,
starvation
attenuates insulin-mediated
glucose disposal,
glycolysis, CHO oxidation, and anaplerosis but enhances
activation of GS.
We thank
the nursing,
dietary,
and technical
staff of the Clinical
Diabetes
and Nutrition
Section,
National
Institute
of Diabetes
and
Digestive
and Kidney
Diseases, for their contributions
and Deb Shilts
for secretarial
assistance.
This research
was supported
by Arizona
Department
of Health
Services Grant 827-OOOOOO-l-0-YD-8358.
Address for reprint
requests:
A. Katz, Dept. of Clinical
Physiology,
Karolinska
Institute,
Karolinska
Hospital,
Box 60500, S-104 01 Stockholm, Sweden.
Received
22 March
1991; accepted
in final
form
16 July
1991.
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