Basal and insul n-mediated carbohydrate metabolism
in human must .e deficient in phosphofructokinase
I
ABRAM
DAVID
KATZ, MARK
K. SPENCER,
STEPHEN
M. MOTT,
RONALD
G. HALLER,
AND
LILLIOJA,
ZHEN YAN,
STEVEN
F. LEWIS
Department
of Kinesiology, University of Illinois, Urbana, Illinois 61801; Clinical Diabetes and Nutrition
Section, National Institute of Diabetes, Digestive, and Kidney Diseases,National Institutes of Health,
Phoenix, Arizona 85016; Departments of Physiology and Neurology, University of Texas Health Science
Center, Dallas 75235; and Veterans Administration Hospital, Dallas, Texas 75216
KATZ, ABRAM, MARK K. SPENCER, STEPHEN LILLIOJA,
ZHEN YAN, DAVID M. MOTT, RONALD G. HALLER, AND STEPHEN F. LEWIS. Basal and insulin-mediated
carbohydrate metabolism in human muscle deficient in phosphofructokinase
1.
Am. J. Physiol. 261 (Endocrinol.
Metab. 24): E473-E478,
1991.-Biopsies
were obtained from the quadriceps
femoris
muscle of two male patients deficient in phosphofructokinase
(PFK) 1. In the basal state the patients had markedly higher
contents of UDP-glucose
(G-fold),
hexose monophosphates
(-7- to 13-fold), inosine monophosphate
(IMP) (-X-fold),
and
fructose 2,6-bisphosphate
(F-2,6-P,;
-6-fold)
than controls.
Fructose 1,6-bisphosphate
was not detectable, and phosphocreatine was lower (33 and 54 mmol/kg dry wt) than in controls
[72 t 4 (SD)]. Patients had normal fasting plasma glucose and
insulin levels and basal glucose turnover rates and responded
normally to a 75-g oral glucose challenge. Patients were also
studied during euglycemic hyperinsulinemia
(-95 mg/dl; 40
and 400 mu. rnB2 min-‘). Whole body glucose disposal rates
were normal during both insulin infusion rates. Biopsies taken
after the 400 mU insulin infusion showed decreases in acetylcarnitine and citrate and increases in the fractional activity of
glycogen synthase. It is suggested that the high basal levels of
F-2,6-P2 are, at least partly, a consequence of the high levels of
fructose 6-phosphate,
which will stimulate flux through PFK2 and inhibit fructose-2,6-bisphosphatase.
The low phosphocreatine and high IMP contents indicate that carbohydrate
availability
is important
for control of high-energy
phosphate
metabolism,
even in the basal state. The insulin-mediated
decreases in acetylcarnitine
and citrate suggest an activation
of the tricarboxylic
acid cycle in skeletal muscle but an absence
of the normal response to replenish these intermediates.
l
tricarboxylic
acid cycle intermediates;
glycogen; hexose monophosphates;
fructose 2,6-bisphosphate;
glucose 1,6-bisphosphate; high-energy
phosphates;
glycogen synthase; glycogen
phosphorylase;
glycogen synthase phosphatase; euglycemic hyperinsulinemia
PHOSPHOFRUCTOKINASE
(PFK) 1 is a key regulatory
enzyme for glycolysis. By inhibiting
PFK and observing
the response of the muscle to different agonists, one can
obtain further information
on the role of PFK in the
regulation of carbohydrate and energy metabolism.
Nature has provided the model with human muscle PFK
deficiency (type VII glycogenosis) (29). Several groups
have used PFK deficiency to study regulatory aspects of
cardiovascular function and muscle metabolism
duri w
0193-1849/91
$1.50
Copyright
exercise (5, 17, 22), but there is limited information
on
the role of PFK and the consequences of the metabolic
adaptations to PFK deficiency (e.g., elevation of hexose
monophosphates)
in hormone-mediated
stimulation
of
carbohydrate metabolism in skeletal muscle. During euglycemic hyperinsulinemia,
skeletal muscle is the major
site of glucose disposal in humans (8). It would therefore
be of interest to determine whether insulin-mediated
glucose disposal is affected by PFK deficiency in muscle,
a situation wherein one might expect a low rate of glucose
utilization
due to inhibition
of hexokinase by high contents of glucose 6-phosphate (G-6-P), as well as an
attenuated increase in carbohydrate oxidation. By determining the metabolic responses of PFK-deficient
muscle
to insulin, new insights into mechanisms of insulin action
may be obtained.
In this report, we provide new information
regarding
the metabolic profile of human skeletal muscle deficient
in PFK, as well as the changes observed during euglycemic hyperinsulinemia.
METHODS
Subjects. Two men, who were diagnosed as PFK deficient based on enzymatic analyses of biopsies from the
quadriceps femoris muscle, participated
in the experiments. The activity of PFK in the muscle of patient 1
was 0.6 mmol. kg wet wt-’ . min-’ and was not detectable
in patient 2 [control = 25.6 t 6.7 (SD)]. Physical characteristics are provided in Table 1. The patients’ health
was assessed by physical examination
and routine hematologic electrocardiograph
and urine tests. The patients were informed of the risks involved in participating
in the experiments before giving voluntary consent. The
experimental
protocol was approved by the ethics committee of the National Institutes of Health.
Experimental design. The patients and controls were
studied during their stay on the metabolic ward (-7
days). The experiments were preceded by at least 3 days
on a weight-maintenance
diet (45% carbohydrate-40%
fat-15% protein). After an overnight fast (-12 h), a twostage euglycemic hyperinsulinemic
clamp was performed
(9). Briefly, at 0600 h and after the patient had voided,
a catheter was placed in an antecubital vein for infusion
of insulin, glucose, and [3-3H]glucose. Another catheter
0 1991 the American
Physiological
Society
E473
E474
TABLE
MUSCLE
METABOLISM
IN
1. Physical characteristics
Patient
Yr
Weight,
kg
Height, cm
Body fat, 5%
2-h OGTT plasma
Glucose, mg/dl
Insulin,
pU/ml
Age,
Values for controls
glucose tolerance
test.
1
Patient
2
Controls
20
56.5
175
14
24
76.0
179
23
24*4
68.8*10.9
17626
12t6
122
26
122
78
97k24
56t37
are means
& SD for
9 subjects.
OGTT,
oral
PFK
DEFICIENCY
neutralized with KHC03. The neutralized extract was
used for fluorometric,
enzymatic (changes in NADPH),
or spectrophotometric
(carnitine) analyses of metabolites (1, 20). The second aliquot was digested in 50 mM
NaOH (8OOC). The extract was neutralized with acetic
acid and assayed spectrophotometrically
for fructose 2,6bisphosphate (F-2,6-P2) using pyrophosphate-dependent
PFK from potato tubers (30). The third aliquot was
digested in 1 M KOH (60°C). The extract was neutralized
with HCl, and glycogen was hydrolyzed enzymatically
(25). Free glucose residues were analyzed enzymatically
(2O), and glycogen was expressed as millimoles of glucosyl
units per kilogram dry weight. A fourth aliquot, when
available, was homogenized in a buffer (133 pl/mg dry
wt) containing 50 mM tris( hydroxymethyl)aminomethane (Tris) l HCl, 10 mM EDTA, and 50 mM 2-mercaptoethanol, pH 7.8, at 4°C. The supernatant (10,000 g for
20 min at 4°C) was assayed for glycogen synthase (GS)
phosphatase as previously described (15). GS phosphatase activity was determined from the change in GS at
low G-6-P concentration
(G&J at 30°C (see below) and
is expressed as millimoles
of UDP-[14C]glucose
incorporated into glycogen per kilogram dry weight per minute
squared.
The second biopsy was also freeze dried, powdered,
and thoroughly mixed. For measurement of GS activity,
the filter paper technique was used (15). 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, 20 mM EDTA, and 130 mM KF,
pH 7.8, at 4°C and then used for assay of GS. GS was
assayed at GSI,, (0.17 mM) and at high G-6-P concentration (7.2 mM) (GShah). UDP-glucose
concentration
was 0.14 mM. Enzyme activity was estimated from the
incorporation of UDP- [14C]glucose into glycogen at 30°C.
The fractional activity is GSlow/GShigh.
Glycogen phosphorylase was assayed on an aliquot of
the 10,000 g supernatant in the direction of glycogen
formation based on the incorporation
of [U-14C]glucose
l-phosphate into glycogen in the absence (phosphorylase
a) or presence (phosphorylase a + b) of 3 mM AMP at
30°C (33). The fractional activity is the ratio of phosphorylase a to phosphorylase a + b.
Plasma glucose was determined with the glucose oxidase method using a Beckman glucose analyzer (Beckman Instruments,
Fullerton, CA), and plasma insulin
was determined by radioimmunoassay,
as modified by
Herbert et al. (10). Plasma free fatty acids were determined enzymatically (21), as was lactate (20), with methods adapted for fluorometry.
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. At 0 min, a
primed continuous infusion of insulin (40 mU rna2.
min-‘) was started and maintained
for 100 min. Thereafter, the insulin infusion rate was increased to 400 mU
m-2
min-’ for an additional 100 min. Infusion of [3-3H] glucose was terminated after the 40 mU insulin infusion.
A variable 20% glucose infusion was performed between
0 and 200 min to maintain the arterialized plasma glucose
concentration at -95 mg/dl. Samples for plasma glucose
were obtained every 5 min throughout the insulin infusion.
Rates of glucose disposal and substrate utilization
(indirect calorimetry) were averaged during the last 40
min of the basal and insulin infusion periods. The rate
of glucose appearance was estimated from the specific
activity of [ 3-3H]glucose in plasma and its rate of infusion using Steele’s non-steady-state
equations (27) during the 40 mU insulin infusion, assuming a glucose
distribution
volume of 100 ml/kg body wt. The rate of
glucose appearance was always lower than the glucose
infusion rate, which indicates that usage of [3-3H]glucose
in the manner that we have to estimate glucose disposal
under the present conditions
(high glucose infusion
rates) is not valid. Glucose disposal was considered to be
equal to the rate of glucose infusion during the 40 and
400 mU insulin infusion. 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 (18).
Biopsies from the lateral aspect of the quadriceps
femoris muscle were obtained with the needle biopsy
technique (2). Briefly, after local anesthesia (10 mg/ml
lidocaine), incisions at biopsy sites on both thighs were
made, and biopsies were obtained before (0 min) and at
the end of the 400 mU insulin infusion clamp. At each
time, two biopsies were taken in rapid succession, the
first for analyses of metabolites
(and in some cases RESULTS AND DISCUSSION
glycogen synthase phosphatase) and the second for enzymes (glycogen synthase and phosphorylase).
Basal. Fasting glycemia and whole body respiratory
exchange ratios were normal (Table 2). In the basal state
Analytical methods. All biopsies were rapidly plunged
the patients had normal rates of glucose turnover.
into liquid N2. The biopsies were stored at -80°C until
Muscle biopsies revealed elevated contents of glycogen
analysis. The samples were freeze dried, dissected free
and diminished contents of
from nonmuscle constituents (blood, fat, connective tis- and hexose monophosphates
fructose 1,6-bisphosphate
and dihydroxyacetone
phossue), and powdered. The powder from the first biopsy
phate (Table 3), all classic indexes of type VII glycogenowas thoroughly mixed and divided into several aliquots.
One aliquot was extracted with 0.5 M perchloric acid and sis (28, 29). Total muscle glucose was quite high at rest.
l
MUSCLE
TABLE
METABOLISM
E475
IN PFK DEFICIENCY
2. Whole body metabolic rates before and after insulin
Insulin
0
40
Patient
Plasma glucose, mg/dl
Plasma insulin, &J/ml
Glucose disposal, mg* kg
FFM-’ . min-’
CHO,,, rng. kg FFM-’ .
min-’
Fat,,, mg kg .FFM-’ . min-’
RER, Vco2/Vo2
88
18
3.36
95
95
5.27
3.78
4.47
400
0
95
1,971
13.0
100
7
2.48
Infusion,
mU
40
1
Patient
99
67
4.34
l
me2 min-’
l
400
0
40
2
400
Controls
96
1,292
14.0
6.14
88t5
17k8
2.62t0.37
9324
116k56
6.36t1.65
94t3
1,669f194
13.3k1.7
1.94t0.61
3.35t0.59
5.0t0.8
0.65
0.29
-0.18
0.81t0.40
0.25kO.28
-0.26kO.24
0.90
0.93
0.99
0.83*
0.97-j. 0.86t0.04
0.93t0.04
1.00t0.04
(0.84)*
Values from controls are means t SD for 9 subjects. * Measured on another day (i.e., not on day of clamp). t Due to technical difficulties
respiratory exchange ratio (RER) was measured with a Delta Trac respiratory analyzing system only at end of high-dose insulin infusion. CHO,,,
carbohydrate oxidation; Fat,,, fat oxidation; FFM, fat-free mass. Fat oxidation is calculated assuming palmitate is being oxidized (mol wt 258).
l
TABLE 3. Glycogen, glycolytic intermediates, and sugar
phosphates in muscle before and after insulin
Insulin
0
Glycogen
Total glucose
UDPG
G-1-P
G-6-P
400
Infusion,
0
mU
l
mB2 - min-’
400
0
Patient 1
Patient 2
Controls
463
476
674
761
379t46
6.6
5.8
4.3
2.3
2.5t0.5
5.2
4.7
3.9
3.9
0.86kO.33
0.88
0.93
1.34
1.66
0.09~0.04
17.1
17.9
15.5
18.8
1.5t0.7
0.072
0.045
0.049
0.034 0.083t0.012
4.1
4.1
3.7
5.3
0.63kO.21
0.01
co.01
co.01
co.01
0.27t0.12
0.054
0.057
0.066
0.076 O.Ollt0.004
0.02
0.03
0.03
0.03
0.17kO.08
0.07
0.04
0.59
0.17
1.2k0.5
G-1,6-P2
F-6-P
F-1,6-P2
F-2,6-P2
DHAP
Glycerol
3-phosphate
Pyruvate
0.09
0.08
0.05
0.04
0.20t0.09
Lactate
2.4
3.7
3.9
1.8
5.5k2.4
Values are given in mmol/kg dry wt and are means t SD for controls,
which represent values for 8 subjects. UDPG, uridine diphosphate
glucose; G, glucose; F, fructose; P, phosphate; Pz, bisphosphate; DHAP,
dihydroxyacetone phosphate. Of metabolites presented, only G-l,6-P2
(after 340 min at 40 mU* m-20min-1), pyruvate, and lactate (after 120
min at 60 mU rnw2amin-‘) increase in response to insulin in controls
(7, 13).
If the content of extracellular water in the biopsy is as
in normals (0.3 l/kg dry wt; see Ref. 11), and we have no
reason to believe otherwise at present (during the processing of the samples, we did not notice any excessive
amounts of dried blood), then the muscle contains significant amounts of free glucose (-5.2 mmol/kg
dry wt
in patient 1 and 2.7 in patient 2) (see Ref. 12 for calculations). In normal subjects, virtually all of the glucose
in the biopsy is confined to the extracellular space (12).
If these estimates of intracellular
glucose are correct,
they could be explained by the elevated levels of G-6-P,
a potent inhibitor of hexokinase (32).
The glucose 1,6-bisphosphate content was within the
normal rangel. UDP-glucose
was increased approximately fivefold in both patients, possibly because of a
block at the level of GS (see below). F-2,6-&, the most
’ While this manuscript was in review, Yamada et al. (Biochem.
Res. Commun.
176: 7-10, 1991) reported that three patients
with type VII glycogenosis had G-l,6-P2 contents in muscle that were
only 20% of that in controls, while the F-2,6-P2 contents were -IO-fold
higher than in controls.
Riophys.
potent activator of PFK-1, was also elevated five- to sixfold. This may be attributed
to the high contents of
fructose 6-phosphate, which will activate PFK-2 and
inhibit fructose-2,6-bisphosphatase
(16).
There were no remarkable differences in the tricarboxylic acid intermediates
(TCAI) and carnitines in the
patients vs. controls in the basal state (Table 4).
Phosphocreatine
was markedly lower, and inosine
monophosphate
was elevated by -15-fold vs. control
values in the basal state (Table 5). To our knowledge,
these are the first analytical determinations
of phosphocreatine and inosine monophosphate
in type VII glycogenosis. Previous studies using nuclear magnetic resonance (NMR) have not found diminished
contents of
phosphocreatine
(5). In fact, using NMR, Chance et al.
(5) found the phosphocreatine
content in one PFKdeficient patient to be -40 mmol/kg
wet wt, which is
equivalent to -172 mmol/kg dry wt, a value that is much
higher than even the total creatine content in our patients as well as the controls. Moreover, Chance et al.
(5) could not detect sugar phosphates in muscle of this
patient at rest, although large increases were detectable
during exercise. These findings lead us to question the
absolute metabolite values obtained by NMR. In erythrocytes from patients with type VII glycogenosis, there
are elevated contents of ADP and AMP (28). If free ADP
and AMP are also elevated in muscle (which is implied
by the low phosphocreatine/creatine
ratio; see Ref. 31),
then these could explain the low phosphocreatine
and
the high inosine monophosphate
contents, since ADP
4. Tricarboxylic acid cycle intermediates
and carnitines in muscle before and after insulin
TABLE
Insulin
0
400
Infusion,
mU - minS2
0
400
l
min-’
0
Patient
1
Patient 2
Controls
Citrate
0.18
0.04
0.35
0.25 0.27t0.07
Fumarate
0.03
0.03
0.06
0.07 0.02&0.02
Malate
0.19
0.23
0.36
0.24 0.33kO.15
Acetylcarnitine
1.29
0.25
0.34
eo.01
0.21t0.15
Free carnitine
10.4
11.5
14.3
13.3
18.3k3.3
Values are given in mmol/kg dry wt and are means & SD for controls,
which represent values for 8 subjects. Of metabolites presented, only
malate increases significantly in response to insulin in controls (after
120 min at 60 mU~m-2*min-‘) (7).
E476
MUSCLE
METABOLISM
5. High-energy phosphates and catabolites
of adenine nucleotides in muscle before and after insulin
TABLE
Insulin
0
400
Infusion,
0
Patient 1
PCr + Cr
PCr
Cr
ATP
ADP
AMP
IMP
Inosine
Adenosine
Hypoxanthine
NAD+
89.4
33.0
56.4
93.2
32.5
60.7
21.7
21.8
3.02
0.14
2.17
0.48
0.01
0.06
2.75
3.12
0.09
2.19
0.55
0.01
0.06
2.63
mU
l
Patient 2
53.9
74.2
21.9
3.22
PFK
DEFICIENCY
6. Enzyme activities in muscle
before and after insulin
TABLE
mS2 - min-’
400
128.1
IN
127.9
52.9
75.0
22.5
3.21
0.10
0.12
1.34
0.75
0.01
0.03
2.51
2.61
0.69
0.01
0.03
2.65
Values are given in mmol/kg
dry wt and are means t
which represent
values from 8 subjects (from Ref. 26).
creatine;
Cr, creatine;
IMP, inosine
monophosphate.
presented
in this table have been shown to change
controls
(40, 60, or 400 mU~m-2~min-‘)
(7, 13, 24, 26).
Insulin
0
0
Controls
112.3k15.1
72.2t3.8
43.223.9
21.7k3.6
3.OlsrO.30
0.14t0.04
0.14kO.06
0.32t0.16
0.01~0.01
0.02~0.01
1.73kO.29
SD for controls,
PCr, phosphoNo metabolites
with insulin
in
would drive the creatine kinase reaction toward a lower
steady-state level of phosphocreatine and since ADP and
AMP would activate AMP deaminase, resulting in inosine monophosphate
formation (14). In this context, it
is interesting to note that, in human skeletal muscle, the
formation of TCAI is heavily dependent on the availability of three-carbon intermediates
derived from glycolysis (25, 26). In type VII glycogenosis, one would expect
this route for formation of TCAI to be blocked. Despite
this, the contents of citrate, malate, and fumarate were
normal. Under these conditions, one could attribute at
least some of the formation of the TCAI to the purine
nucleotide cycle (19). The high inosine monophosphate
contents are consistent with this hypothesis. Thus high
free ADP and AMP contents may be viewed as adaptations necessary to maintain adequate levels of TCAI for
mitochondrial
ATP synthesis in type VII glycogenosis.
Total tissue adenine nucleotides, as well as their catabolites (except for inosine monophosphate),
appeared
to be normal. NAD+ was slightly increased. This may be
due to increased synthesis via increased flux through the
pentose shunt (due to high G-6-P).
The fractional activity of GS was lower than normal
in the basal state, whereas the fractional activity of
phosphorylase tended to be higher (Table 6). The lower
GS fractional activity is in contrast to an earlier report
based on one patient in whom a markedly higher fractional activity was reported (vs. 2 controls) (23). The low
fractional activity of GS, despite apparently normal activities of GS phosphatase may be attributed, at least in
part, to the elevated contents of glycogen, which may
shield the phosphorylated sites of GS from GS phosphatase (6). Alternatively,
the activity of a protein kinase(s)
(e.g., adenosine 3’,5’-cyclic
monophosphate-dependent
protein kinase) may be elevated, and this could explain
the altered fractional activities of GS and phosphorylase.
Glucose tolerance and insulin sensitivity. The two patients were found to have normal glucose tolerance
(Table 1). The plasma glucose results are comparable to
those reported earlier by Tarui et al. (28). Both patients
had normal glucose disposal rates in response to insulin
400
Patient 1
GSow
GShigh
G&nv/GShigh
Phos a
Phos a+b
Phos a/Phos
Protein
GS phosphatase
0.14
1.37
0.10
2.11
5.85
0.36
29
a+b
69
148
0.20
350
ND
366
0.19
565
, 0.333
Infusion,
0
mU - m-’ - min-’
400
Patient 2
0.30
1.77
0.17
38
192
0.20
414
0.364
0.95
3.41
0.28
58
274
0.21
616
ND
0
Controls
0.80t0.34
1.8920.35
0.42kO. 14
16klO
199t70
0.09kO.06
621k36
0.253t,O.O40
Values are given in mmol kg dry wt-’ l rein-’ at 30°C (GS phosphatase is in mmol. kg dry wt-’ rein-‘)
and are means t SD for controls,
which represent
values for 7 or 8 subjects. GS,,,, low G-6-P concentration; GShiah, high G-6-P concentration;
GS, glycogen
synthase;
Phos,
phosphorylase.
ND, not determined.
l
l
infusion (Table 2). We should note that we have not
performed
euglycemic hyperinsulinemic
clamps with
muscle biopsies on normal subjects in exactly the same
manner (i.e., duration and dose of insulin infusion, as
well as the order of the doses) as in the two patients.
However, we have performed euglycemic hyperinsulinemic clamps with biopsies on normals at 40-400 mU
rnB2 l rein-1 for 120-340 min. Therefore, the noteworthy
results on these patients will be compared with the data
from these studies (see below).
In view of the finding that, during euglycemic hyperinsulinemia,
-90% of the infused glucose is taken up by
skeletal muscle (8), we would have predicted a marked
insulin resistance. This is because the high G-6-P content is expected to inhibit hexokinase (32). Indeed, we
have, on several occasions, demonstrated
that glucose
utilization in human muscle is inhibited under conditions
where G-6-P is elevated (11, 24). The following explanations for the normal rates of glucose disposal in these
patients may be considered. One is that hexokinase in
these patients may not be as sensitive to G-6-P as is
hexokinase of normal subjects. Alternatively,
most of the
glucose utilization
during euglycemic hyperinsulinemia
in type VII glycogenosis is not accounted for by skeletal
muscle. Balance studies are needed to determine which
tissue(s) is responsible for the glucose utilization.
An unexpected finding was the normal rate of carbohydrate oxidation inpatient 1. Again, balance studies are
needed to determine which tissue(s) is responsible for
the glucose oxidation.
An interesting
finding was that insulin resulted in
consistent decreases in citrate and acetylcarnitine.
In
normal subjects, we have never observed a decrease in
citrate during euglycemic hyperinsulinemia
at infusion
rates of 40 (7,13,24), 60 (7), or 400 mU~m-2~min-1
(13),
nor have we observed any change in acetylcarnitine
during a 60 mU rnD2. min-’ insulin clamp. The results
from the PFK-deficient
patients indicate an insulinmediated activation of the TCA cycle in muscle, as has
been shown in the isolated rat diaphragm (3), with an
inadequate compensatory activation of anaplerotic processes to replenish the TCAI. In normals, insulin stimulates glycolysis (7,24), thereby providing adeauate three-
MUSCLE
METABOLISM
carbon intermediates
for replenishing
the TCAI, such
that no decrease is observed in citrate, malate, or fumarate (at 40 mu. mD2 gmin-‘) (26). In fact, if the insulin
infusion rate is sufficiently high, e.g., 60 mu. mB2 min-‘,
then a significant increase in malate is observed (7).
Insulin resulted in an increase in the fractional activity
of GS but no change in the fractional activity of phosphorylase. These findings are in agreement with those
observed under comparable conditions in normals (33)
and further demonstrate an insulin-mediated
effect on
skeletal muscle. The data on GS phosphatase are incomplete.
The antilipolytic
effect of insulin in patient 1 was
normal, as judged by the steady decrease in plasma free
fatty acids during the 40 mU insulin clamp (basal = 213
PM; end of clamp = 77 PM) (control = 252 t 74 to 122
t 102) (24). A steady decrease was also observed in his
plasma lactate values under these conditions (basal =
0.95 mM; end of low-dose clamp = 0.58 mM). Normally,
plasma lactate increases, and splanchnic lactate uptake
decreases during euglycemic hyperinsulinemia
(4). If
splanchnic metabolism
during euglycemic hyperinsulinemia is not affected by muscle PFK deficiency, then
the decrease in plasma lactate may be attributed to an
increased lactate uptake by peripheral tissue(s), possibly
skeletal muscle. This may explain at least part of the
increase in carbohydrate oxidation during euglycemic
hyperinsulinemia.
This interpretation
is consistent with
the observation that administration
of oxidizable substrate in the form of lactate can enhance contractionmediated increases in muscle oxygen consumption (17).
In summary, we have described some new anomalies
in the metabolic profile of human skeletal muscle deficient in PFK in the basal state and after administration
of insulin. We have also demonstrated
normal glucose
tolerance and insulin-mediated
whole body glucose disposal, but the tissue(s) responsible for the glucose utilization remains to be determined.
l
We thank the nursing and dietary
staff, Karen Stone (for analyses
of glycogen synthase
and phosphorylase)
of the Clinical
Diabetes
and
Nutrition
Section of the National
Institute
of Diabetes
and Digestive
and Kidney
Diseases, and Deb Shilts for secretarial
assistance.
This research was supported
by National
Heart,
Lung, and Blood
Institute
(NHLBI)
Grant HL-06296,
the Harry
S. Moss Heart Center,
the Department
of Veterans
Affairs,
and the Muscular
Dystrophy
Association.
S. F. Lewis is the recipient
of NHLBI
Research
Career
Development
Award HL-01581.
Present address of S. F. Lewis: Dept. of Health
Sciences,
Boston
Univ., 635 Commonwealth
Ave., Boston, MA 02215.
Address for reprint
requests: A. Katz, Dept. of Clinical
Physiology,
Karolinska
Institute,
Karolinska
Hospital,
Box 60500, S-104 01 Stockholm, Sweden.
Received
19 February
1991; accepted
in final
form
12 June
1991.
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