From the
The isolated working rat heart was adapted for simultaneous
determination of glycogen synthesis and degradation using a dual
isotope technique. After prelabeling of glycogen with
[U-
The experimental reports regarding simultaneous glycogen
synthesis and degradation, mostly pertaining to liver, are conflicting.
Glycogen turnover is indicated in the liver by
The specific aim of the
present study, initially, was to test the hypothesis that obligatory
interconversion of glucose through the glycogen pool occurs before
glycolysis, serving as a mechanism to maintain glycogen reserves. Thus,
[5-
Because of uncertainty regarding the
appropriate specific radioactivity of glycogen, which should be used to
calculate rates of glycogenolysis, and other possible sources of error,
perfusions were conducted to compare the change in glycogen content
between hearts that were frozen at the beginning and end of the study
period, to the change in glycogen content predicted from the calculated
rates of glycogenolysis and glycogen synthesis. Good agreement was
obtained if it was assumed that the specific radioactivity of the
glycogen being mobilized during the study period is the same as the
average specific activity of all the glycogen. This result is not
consistent with the existence of molecular order in the synthesis and
degradation of glycogen
(11, 12) . When we analyzed the
specific radioactivity of the glycogen remaining after varying degrees
of glycogenolysis, the results were also inconsistent with absolutely
selective removal of the most recently synthesized glycogen, although
partial temporal selectivity was still discernable.
The perfusion protocol is shown in
Fig. 1A. Hearts were perfused for the first 15 min
without substrate to deplete endogenous substrates. At this time the
perfusate (200 ml) was supplemented with 5 m
M glucose, 10
m
M lactate, and 0.05 µCi/ml
[U-
Hearts, stored at -70 °C, were weighed,
powdered under liquid N
Fig. 1A depicts the perfusion protocol and
corresponding contractile performance (Fig. 1 B,
hydraulic power) in 3 of the four groups of perfusions. A fourth group
was freeze-clamped at 45 min for determination of glycogen content and
specific radioactivity at the beginning of the study period.
Contractile performance in this group is omitted from the figure for
clarity. The four groups were subjected to the same perfusion
conditions until 45 min of the protocol to achieve the same glycogen
content and specific radioactivity, and did not differ in contractile
performance until that time. There was rapid decline in power during
the first 15 min of the perfusions as a consequence of the omission of
substrate from the perfusate. Contractile performance was largely
restored upon introduction of substrates (glucose plus lactate) at 15
min. Perfusions were continued for an additional 25 min to allow the
resynthesis of glycogen from [U-
Hearts freeze-clamped at 45 min of the protocol
contained 89.3 ± 3.4 µmol/g, dry weight, total glycogen, and
50.4 ± 7.5 µmol/g, dry weight, of
[
Fig. 2
shows the time course of release of
Glycolytic fluxes are presented in ,
and include flux from exogenous glucose (
In an attempt to better quantify the simultaneous rates of
glycogen synthesis and glycogenolysis, synthesis was calculated from
net tritium incorporation by correction for detrition resulting from
simultaneous degradation. Degradation, in turn, was corrected for
isotopic dilution by unlabeled glycogen. Because of the necessary
simplifying assumptions in applying these corrections (discussed
below), it became important to check the balance of glycogen during the
study period as predicted from the derived rates of synthesis and
degradation, as compared to the change in glycogen content between
hearts frozen at the beginning and at the end of the study period.
Net incorporation of [5-
The mathematical model used to correct
synthesis for simultaneous glycogenolysis makes three assumptions (see
``Appendix''): that glycogen synthesis and degradation occur
at a constant rate and that concurrent glycogenolysis does not
differentiate between newly incorporated
[
The rates of glycogenolysis
presented in account for isotopic dilution by unlabeled
glycogen. They were calculated assuming that the specific radioactivity
averaged over all the glycogen is representative of the glycogen
converted to
The reasonably
good agreement between measured and predicted glycogen balances
illustrated above supports the assumption of uniform isotopic dilution
of [
When a plot was constructed of the
percent enrichment of the remaining glycogen versus the amount
of remaining glycogen (Fig. 4) the result was not consistent with
absolutely selective removal of the more recently incorporated
[
Values are the mean ± S.E.
in µmol/g, dry weight. Changes in total and
[
We are grateful to Gary D. Lopaschuk for helpful
discussion and Sonya G. Carmical for expert secretarial assistance. APPENDIX
The rate of glycogen synthesis was calculated from net
[5-
On-line formulae not verified for accuracy If G
On-line formulae not verified for accuracy Substituting for G
On-line formulae not verified for accuracy The value of C (the constant of integration) was
determined from the initial condition that at t = 0,
N = 0:
On-line formulae not verified for accuracy The following experimental values were substituted in the case of
perfusions conducted in the absence of added hormones:
V
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
C]glucose, glycogenolysis was determined
continuously from the washout of
CO
plus
[
C]lactate. Glycogen synthesis was determined
during the same period from incorporation of
[5-
H]glucose. In the absence of added hormones,
hearts were predominantly glycogenolytic (1.5 µmol/min/g, dry
weight), and there was simultaneous synthesis (11% of the rate of
glycogenolysis). The percentage of glucose taken up by the heart that
could traverse the glycogen pool as a consequence of glycogen turnover
was minor (5%). Insulin (10 milliunits/ml) predictably stimulated
glycogen synthesis (3.6-fold) and nearly abolished glycogenolysis.
Addition of glucagon (1 µg/ml) increased contractile performance
and initially stimulated glycogenolysis (3.8-fold) until glycogen was
largely depleted. Net tritium incorporation was unaffected by glucagon.
Both hormones stimulated glycolytic flux from exogenous glucose
(
H
O from [5-
H]glucose) as
well as total glycolytic flux (
H
O plus
glycogenolysis). The initial stimulation in total glycolytic flux with
glucagon was largely from glycogen, explaining the lag in stimulation
from exogenous glucose. The relationship between the specific
radioactivity and amount of glycogen remaining after different degrees
of glycogenolysis suggests that the preference of glycogenolysis for
newly synthesized glycogen is only partial.
C NMR
studies which show a decline in the
C resonance from
glycogen previously labeled from [
C]glucose,
despite continued glycogen synthesis
(1, 2, 3) .
Other isotopic studies either support
(4, 5) or refute
(6, 7) the occurrence of hepatic glycogen futile
cycling. There is a single report of significant glycogen turnover in
isolated skeletal muscle, and changes upon hormonal addition
(8) . Finally, in a single study, glycogen turnover was
investigated in rat heart by
C NMR in a manner similar to
that described above for the liver
(9) . The persistence of
[
C]glycogen during the ``chase,'' when
[
C]glucose replaced
[
C]glucose indicated that no turnover of the
newly synthesized glycogen had occurred.
H]glucose was chosen because this isotope is
retained upon incorporation into glycogen, and can also be used to
trace glycolytic flux from exogenous glucose by release of
H
O, which occurs during triose interconversion
(10) . It was found that glycolytic flux exceeds, by a factor of
15-20, the potential interconversion of glucose through the
glycogen pool as a consequence of glycogen turnover. In the present
study, the majority of glucose entering the cell bypassed glycogen.
Although our initial hypothesis seems unlikely, a small amount of
glycogen turnover was detected, at least in the absence of added
hormones. Addition of insulin stabilized recently synthesized glycogen
by blocking glycogenolysis. This result is therefore in agreement with
the
C NMR study of rat heart mentioned above
(9) since insulin was included throughout the NMR study.
Glucagon was investigated because of its glycogenolytic action.
However, the influence of glucagon on glycogen synthesis and,
therefore, on glycogen turnover was more difficult to interpret because
rates of glycogen degradation with glucagon addition were not constant
during the study period, precluding calculation of the rate of
synthesis after correction for simultaneous tritium removal by
concurrent glycogenolysis.
Materials
D-[U-C]Glucose
was obtained from ICN Biomedicals (Costa Mesa, CA).
D-[5-
H]Glucose was from Amersham Corp.
Enzymes for metabolite assays were from Boehringer Mannheim. Other
chemicals were from Sigma.
Heart Perfusions
Hearts from 300-350-g
chow-fed male Sprague-Dawley rats (Harlan, Indianapolis, IN) were
perfused in the working heart apparatus
(13) as described
previously
(14) , with modification to permit collection of
CO
as described below. Rats were anesthetized
with sodium pentobarbital (100 mg/kg), heparin (100 units) injected
into the inferior vena cava, and the hearts were quickly excised and
placed in ice-cold buffer. The aorta was cannulated and retrograde
(Langendorff) perfusion begun with warm (37 °C) buffer while
cannulating the opening to the left atrium. The perfusate consisted of
Krebs-Henseleit buffer equilibrated with 95% O
, 5%
CO
. The CaCl
concentration was 1.25
m
M. Glucose (5 m
M) was present during the initial
retrograde perfusion and the remainder of the perfusion with the
exception of the first 15 min. Hearts were switched to the
recirculating mode after 5 min of retrograde perfusion by opening the
line to the left atrium, and switching the aortic line from the
Langendorff reservoir to an overflow chamber within the recirculating
circuit. The fluid level in this chamber was maintained 82 cm above the
heart (aortic afterload). The atrial filling pressure was 15 cm
H
O.
C]glucose by injecting 1 ml of 2
M
sodium
L-lactate, 1
M glucose, and 10 µCi of
[U-
C]glucose into the stirred reservoir, and the
perfusions were continued for 25 min to allow the return of contractile
performance and synthesis of [
C]glycogen. This
time period (25 min) was chosen based on the results of a preliminary
experiment in which [2-
H]glucose was included to
determine glucose uptake and phosphorylation by release of
H
O
(15) ; glucose uptake was largely
suppressed by lactate within 20 min after the addition of glucose plus
lactate, indicating little or no further glycogen synthesis. Perfusions
were switched to a non-recirculating mode between 40 and 45 min of the
protocol to remove [U-
C]glucose and lactate from
the apparatus and the heart, and the apparatus was recharged with 200
ml of fresh perfusate containing [5-
H]glucose (5
m
M, 0.05 µCi/ml) upon restarting the recirculating mode.
The [U-
C]glucose in the perfusate was reduced to
less than 0.5% of the concentration present before the
non-recirculating interval. Samples of perfusate (2.5 ml) were
collected from the reservoir (equipped with a magnetic stir bar) at
5-min intervals by way of a gas tight port for determination of
CO
,
H
O, and
[
C]lactate. Accumulation of metabolites in the
perfusate was calculated from the concentration multiplied by the
volume of perfusate remaining at the time of sample collection,
accounting for the decrease in volume resulting from repeated sampling.
Perfusions were terminated after the subsequent 30-min study period by
freezing the hearts on their cannulas with the use of aluminum tongs
cooled in liquid N
. In one set of perfusions, hearts were
freeze-clamped at the end of the non-recirculating period (45 min) for
determination of glycogen content and
C enrichment of the
glycogen at the beginning of the study period. By inclusion of
[5-
H]glucose in the non-recirculating perfusate
(5 m
M, 0.05 µCi/ml), the same perfusions (freeze-clamped
at 45 min) were used to determine contamination, if any, of the
glycogen, after extraction and purification, from
[5-
H]glucose present in the perfusate, to be used
as a blank correction in the determination of tritium incorporation
resulting specifically from glycogen synthesis. There were no
detectable tritium counts associated with the glycogen in these
perfusions, and a blank correction for
C and
H
incorporation into glycogen resulting from contamination by the
perfusate was deemed unnecessary.
Figure 1:
Perfusion protocol
and contractile performance in the isolated working rat heart. The
perfusion protocol ( A) is lined up with the corresponding
contractile performance ( B). Values are the mean ± S.E.
in milliwatts for four or five perfusions in each group. * p < 0.05 compared to controls (no addition at 45 min). The
symbols are: (), control; (
), insulin (10 milliunits/ml)
added at 45 min; (
), glucagon (1 µg/ml) added at 45
min.
The apparatus was rendered gas
tight to permit quantitative collection of CO
and includes
CO
accumulating in the
perfusate as well as
CO
exhausted from the
oxygenator. In the latter case, the exhaust from the oxygenator (2
liters/min) was passed through a water trap, then directed to a
CO
trap contained within a scintillation vial with the use
of a gas dispersion tube. The exhausted
CO
was
collected over 5-min intervals by bubbling through 5 ml of 0.3
M benzethonium hydroxide in methanol; it was then subjected to
scintillation counting after addition of 10 ml of scintillation mixture
(Ultima Gold, Packard, Meriden, CT). The content of
CO
in the perfusate was determined by trapping
CO
liberated upon acidifying perfusate samples (0.5 ml),
collected at 5-min intervals. This was accomplished by transferring the
sample to a 1.5-ml microcentrifuge tube, placing the tube within a
scintillation vial containing 1 ml of 1
M benzethonium
hydroxide in methanol, and sealing the vial with a serum cap before
injecting 0.1 ml of 60% perchloric acid into the perfusate. Collection
of CO
was continued overnight with gentle agitation, the
centrifuge tube removed, and the vial processed for scintillation
counting as above. Determinations were corrected for background
radioactivity by trapping exhausted CO
for 5 min before
cannulating the heart and by trapping CO
from perfusate
taken before cannulating the heart. In order to prevent the formation
of pressure gradients within the apparatus once it was rendered gas
tight, which would alter the pump action of the heart, air spaces
within the apparatus were interconnected by vent tubing. The
interconnected air spaces consisted of the oxygenator, heart chamber,
aortic overflow chamber, a graduated chamber for determination of
coronary flow, and the reservoir at the base of the apparatus.
Quantitative recovery of
CO
was verified in a
preliminary experiment by injecting a bolus of
[
C]NaHCO
(2 µCi), which was
transferred from the perfusate into the gas phase over time.
Analytical Procedures
Tritiated water was
determined in samples of perfusate (0.5 ml) after passing through 2-ml
columns of AG1-X8 resin, hydroxide form (Bio-Rad) essentially as
described previously
(16) . The H
O
passing through the columns was collected in two 3-ml fractions for
determination of radioactivity after addition of 10 ml of scintillation
mixture. The columns were then used to determine
[
C]lactate on the same samples, adapted from a
previously described method
(17) : after removal of
[5-
H]glucose by washing with 500 ml of water, the
[
C]lactate (and other anions) remaining on the
column was eluted with 0.2
M sodium acetate, and collected in
four 4-ml fractions for determination of radioactivity after addition
of 10 ml of scintillation mixture. A preliminary experiment was
conducted to verify the quantitative determination of a known amount of
authentic [
C]lactate in a sample of perfusate
containing a large excess of [5-
H]glucose.
Radioactive lactate determinations were corrected for a blank obtained
with the use of a sample of perfusate collected at 46 min of each
perfusion.
, and a portion of the tissue powder
taken for dry weight determination. Glycogen was determined on the
powdered tissue as glucose
(18) following digestion with KOH,
repeated ethanol precipitation, and digestion with amyloglucosidase
(19) . A portion of the glycogen digest was taken for
scintillation counting to determine net
H and
C incorporation. Quench correction and simultaneous
determination of
H and
C by spectral index
analysis were performed by routines supplied with the instrument
(Packard 1900 TR). Data are expressed as the mean ± S.E.
Metabolic rates were determined by least squares linear regression of
plots of total metabolite accumulation versus perfusion time.
Statistical comparison was by way of analysis of variance with post hoc
comparison by Newman-Keuls multisample test. p < 0.05 was
considered significant.
C]glucose, then
the hearts subjected to a non-recirculating interval (40-45 min)
with fresh perfusate in order to wash lactate and
[U-
C]glucose from the apparatus and the heart.
The recirculating perfusion was reestablished between 45 and 75 min
with fresh perfusate containing [5-
H]glucose in
place of lactate and [U-
C]glucose. In the
absence of added hormones, there was a gradual, but insignificant,
decline in contractile performance during this 30-min interval.
Addition of insulin at the beginning of this interval (45 min) slightly
improved performance, although the difference was not significant
relative to controls. Addition of glucagon at 45 min resulted in
pronounced stimulation in contractile performance (70% relative to the
value at 40 min).
C]glycogen ( n = 6), calculated
from the
C content of the glycogen after extraction and
purification, and the specific radioactivity of the
[U-
C]glucose. The enrichment of the glycogen,
relative to the [U-
C]glucose precursor was 55.8
± 6.7%. The difference between the total glycogen and
[
C]glycogen, 38.9 ± 5.7 µmol/g, dry
weight, is the extrapolated glycogen content at the end of 15 min of
perfusion without substrate, neglecting the small change between 40 and
45 min. We previously found values of 82 and 127 µmol/g, dry
weight, for rat heart in vivo and after 15 min of perfusion in
the presence of glucose, respectively
(14) . Therefore, in the
present study, 43 µmol/g, dry weight, was broken down during the
first 15 min of perfusion as a consequence of omitting substrates.
Hearts from fed rats otherwise synthesize glycogen during this 15-min
interval, if glucose is not omitted
(14) . The subsequent net
incorporation of 50.4 µmol/g, dry weight, of
[
C]glycogen between 15 and 45 min is the amount
available during the study period (45-75 min) for the measurement
of glycogenolysis as release of
CO
plus
[
C]lactate. This value for net
C
incorporation measured directly in hearts freeze-clamped at 45 min
compares favorably with the amount calculated in the other groups from
data presented in . The calculated estimate in the other
groups is the sum of [
C]glycogen remaining at 75
min plus the amount released as
CO
plus
[
C]lactate between 45 and 75 min (43 ± 6,
60 ± 7, and 43 ± 2 µmol/g, dry weight, for controls,
insulin, and glucagon groups, respectively), indicating that similar
levels of glycogen enrichment had been achieved in the different
groups.
CO
plus [
C]lactate.
Values were calculated in terms of glycosyl units, using the specific
radioactivity of the [U-
C]glucose included to
prelabel the glycogen, and will tend to underestimate the true rate of
glycogenolysis to the extent that isotopic dilution by unlabeled
glycogen will occur (see below). In the absence of added hormones,
glycogen breakdown was essentially linear for the 30-min study period.
Insulin addition (10 milliunits/ml) at the beginning of this period
resulted in a time-dependant decrease in the rate of release of
CO
plus [
C]lactate,
attaining a value of 0.06 ± 0.02 µmol/min/g, dry weight,
during the second half of the study period (the value presented in
Table II was calculated over the entire 30 min). Insulin preserved
[
C]glycogen indicating that the diminished
glycogenolysis was not a consequence of glycogen depletion
(). Addition of glucagon (1 µg/ml) initially stimulated
glycogenolysis (3.8-fold). The subsequent decline in glycogen breakdown
after 10 min of glucagon stimulation was the result of glycogen
depletion (see Table I, final values for total and
[
C]glycogen content). Therefore, the rate of
glucagon-stimulated glycogenolysis in is the initial
rate, determined between 45 and 55 min. The percentage of
C released from glycogen in the form of
CO
plus [
C]lactate that
appeared in the form of [
C]lactate was constant
during the study period, and averaged 14 ± 2% in the controls.
This was increased ( p < 0.05) by both insulin and glucagon,
averaging 32 ± 5% and 29 ± 3%, respectively.
Figure 2:
Time
course of release of CO
plus
[
C]lactate during the study period. Hearts were
subjected to the [
C]glycogen loading protocol of
Fig. 1, and after removing [U-
C]glucose,
monitored for release of
CO
plus
[
C]lactate between 45 and 75 min. Values were
calculated based on the specific radioactivity of the
[U-
C]glucose, and are not corrected for isotopic
dilution by unlabeled glycogen. Values are the mean ± S.E. in
µmol/g, dry weight, for four or five perfusions in each group.
* p < 0.05 compared to controls. The symbols are: (
),
control (no addition at 45 min); (
), insulin (10 milliunits/ml)
added at 45 min; (
), glucagon (1 µg/ml) added at 45
min.
presents rates of glycogen synthesis, glycogenolysis, and
glycolytic fluxes. In the controls, in which glycogenolysis was
continuous during the study period (Fig. 2), glycogen synthesis
(11% of the rate of glycogenolysis) must have occurred concurrent with
glycogenolysis. The measured rate of net tritium incorporation averaged
over the entire 30-min study period in the controls (0.12 ± 0.03
µmol/min/g, dry weight) is 30% less then the calculated rate of
glycogen synthesis presented in the table because of correction for
detrition resulting from simultaneous glycogenolysis (see
``Appendix''). In the presence of insulin, glycogenolysis was
inhibited to the extent that detrition by concurrent glycogenolysis was
neglected, so that the rate of net tritium incorporation was used as
the best estimate of the true rate of glycogen synthesis. Glycogen
synthesis was stimulated 3.6-fold by insulin. Glucagon did not
influence net tritium incorporation relative to controls. The rate of
net tritium incorporation averaged over 30 min is presented in
as a minimal estimate of glycogen synthesis in the
presence of glucagon, because the value is not corrected for concurrent
glycogenolysis.
H
O
production from [5-
H]glucose) and total
glycolytic flux, which includes the contribution by glycogenolysis.
Both insulin and glucagon stimulated glycolytic flux from exogenous
glucose as well as the total glycolytic flux. Fig. 3shows the
time course of glycolytic fluxes (total and from exogeneous glucose) as
well as glycogenolysis on the same scale, following stimulation with
glucagon. Upon addition of glucagon, there was a lag in the stimulation
of glycolytic flux from exogenous glucose. The lag occurred during a
period of rapid glycogenolysis, explaining the constancy of total
glycolytic flux. Once glycogen was depleted, the glucagon stimulated
rates of glycolysis (both total and from exogenous glucose) were
comparable to the rates obtained after insulin stimulation (Table II).
Since the principal fates of glucose in the heart are either glycogen
synthesis or glycolysis, glucose uptake by the heart can be estimated
from the sum of glycogen synthesis plus glycolytic flux from exogenous
glucose. Rates of glycogen synthesis were small compared to glycolytic
flux, so that the rates of glycolytic flux from exogenous glucose
presented in the table will only slightly underestimate glucose uptake.
Approximately 4.5 and 7.5% of the glucose taken up by the heart was
directed toward glycogen synthesis in the absence and presence of
insulin, respectively, and this is the maximum percentage of glucose
taken up by the heart that could traverse the glycogen pool prior to
glycolysis as a consequence of glycogen turnover.
Figure 3:
Time
course of glycolytic flux from exogenous glucose, glycogen and total
flux (glucose plus glycogen) following stimulation with glucagon. The
figure shows the glucagon-treated hearts described in the legends to
Figs. 1 and 2. [5-H]Glucose (5 m
M, 0.05
µCi/ml) was included along with glucagon (1 µg/ml) at 45 min
for determination of glycolytic flux from exogenous glucose (
)
by release of
H
O. Glycogen breakdown (
)
was determined from the release of
CO
plus
[
C]lactate as described in the legend to Fig. 2,
after correction for isotopic dilution from unlabeled glycogen as
described in the text. Total glycolytic flux (
) is the value from
glucose plus glycogen. Values are the mean ± S.E. ( n = 4) in µmol/g, dry weight.
H]glucose into
glycogen, as measured in the present study, will tend to underestimate
total tritium incorporation and, therefore, to provide a minimal
estimate of glycogen synthesis to the extent that simultaneous
degradation results in tritium removal. In those perfusions where
insulin was added, net tritium incorporation should provide a true
estimate of glycogen synthesis since glycogenolysis was essentially
abolished by the addition of insulin. In the case of perfusions
conducted in the absence of added hormones, it was possible to estimate
the degree to which glycogen synthesis was underestimated by net
tritium incorporation from knowledge of the rate of glycogenolysis in
the same perfusions. The extent of net tritium incorporation
underestimated the calculated rate of glycogen synthesis by
approximately 30% ( i.e. 0.12 versus 0.17
µmol/min/g, dry weight).
H]glycosyl residues and the remainder of the
glycogen molecule. The assumption of constant glycogenolysis was
established in the control group but not in the other groups
(Fig. 2). Regarding the third assumption, glycogenolysis appears
to be only partially selective for the more recently synthesized
[
C]glycogen (see below). If glycogenolysis is
highly selective for the most recently incorporated
[5-
H]glycosyl residues, then the degree to which
net [5-
H]glucose incorporation underestimates the
actual rate of glycogen synthesis will be larger than 30%, and the
extent of glycogen turnover in the control perfusions will be larger
than estimated in the present study. Glucagon did not influence net
tritium incorporation relative to controls. However, because of
extensive glycogenolysis, it is likely that net incorporation in the
presence of glucagon will underestimate glycogen synthesis to a greater
extent than in the absence of glucagon.
CO
plus
[
C]lactate during the study period. In other
words, it was assumed that the glycogen was degraded randomly, without
regard for the temporal order of incorporation of
[U-
C]glycosyl residues, so that a fixed value
for the specific radioactivity of the glycogen (55.8% relative to the
[U-
C]glucose precursor) could be applied. The
justification for calculating glycogenolysis in this manner was
derived, first, from a consideration of the balance of total glycogen
during the 30-min study period in relation to the change in total
glycogen predicted from the measured rates of synthesis and
degradation. Second, the pattern of glycogen specific radioactivity at
the end of the perfusions suggested that
[
C]glycosyl residues were partially randomized
with respect to subsequent glycogenolysis (see below). Regarding the
glycogen balance, in the case of the control group, new glycogen
synthesis amounted to 5.1 µmol/g, dry weight (30 min
0.17
µmol/min/g, dry weight). Assuming random degradation, the total
degradation will be the total release of
CO
plus [
C]lactate calculated at the specific
activity of the [U-
C]glucose (24.6 ± 2.5
µmol/g, dry weight, ) divided by the fractional
C enrichment of glycogen (0.558), or 44.1 µmol/g, dry
weight. The predicted balance of total glycogen is synthesis minus
degradation, or 5.1 - 44.1 = -39.0 µmol/g, dry
weight (the value calculated from the individual perfusions was
-39.0 ± 4.3). This value agrees favorably with the
measured glycogen balance of -36.5 ± 4.4, obtained from
the change in total glycogen content between hearts freeze-clamped at
the beginning and end of the study period (). In the case
of perfusions in the presence of insulin, the corresponding predicted
and measured glycogen balances were +5.6 ± 4.9 and
+16.8 ± 5.9 µmol/g, dry weight, respectively. The lack
of agreement in the presence of insulin may reflect overestimation of
the low rates of glycogenolysis because of small residual amounts of
C-labeled metabolites other than glycogen contributing to
the release of
CO
plus
[
C]lactate. Indeed, comparison of the amount of
incorporation of [5-
H]glucose to the glycogen
balance measured by the change in content (18.5 ± 4.3 versus 16.8 ± 5.9 µmol/g, dry weight, respectively,
) suggests that there was virtually no glycogenolysis in
the presence of insulin. Only a minimum estimate of glycogen synthesis
is available for glucagon treated perfusions. However, error in the
rate of synthesis does not appreciably effect the calculation of
glycogen balance from synthesis minus degradation because synthesis is
comparatively small in the presence of this hormone. Assuming a
synthesis rate of 0.17 µmol/min/g, dry weight, in the presence of
glucagon ( i.e. the same as in control perfusions) the balance
of total glycogen in the glucagon containing perfusions predicted from
synthesis minus degradation (5.1 - 69.4) is -64.3 ±
4.3, which compares favorably to the measured value of -72.1
± 6.5 µmol/g, dry weight ().
C]glycosyl residues by preexisting glycogen,
that was made to calculate rates of glycogenolysis. In reality, because
of the evidence in favor of molecular order in the synthesis and
degradation of glycogen
(11, 12) , it seems more likely
that the hearts exhibited partial temporal selectivity of
glycogenolysis (see below), although the effect was obscured by a
combination of biological variability and high
C
enrichment of the glycogen. The protocol was designed to maximize the
fractional enrichment of the glycogen, while allowing for good return
of contractile performance, to reduce the uncertainty in specific
radioactivity of the glycogen being mobilized at any given time during
the time course of the study period. To this end, endogenous glycogen
was depleted by perfusion without substrate prior to stimulating
glycogen synthesis from [U-
C]glucose by the
addition of lactate
(20) .
C]glycogen, although glycogenolysis was not
completely random. The lower line in the figure is the hypothetical
result if phosphorylase specifically removed
[
C]glycogen before removing the older, unlabeled
glycogen. Since 56% of the glycogen was synthesized at the specific
activity of the [U-
C]glucose, selective removal
of this 56% would result in 44% of the glycogen remaining (39
µmol/g, dry weight), and the remaining glycogen would be
unenriched. In contrast, the horizontal line in Fig. 4is the
result expected if glycogenolysis occurred at random, with complete
disregard for the temporal order of incorporation. The regression line
through experimentally derived values suggests that glycogenolysis was
substantially random with respect to the age of glycosyl residues being
removed, although partial temporal selectivity is discernable.
Figure 4:
Enrichment of glycogen versus amount of glycogen remaining. The C enrichment of the
glycogen remaining after different degrees of glycogenolysis was
determined relative to the amount of glycogen remaining in hearts
subjected to the protocol depicted in Fig. 1. The enrichment was
calculated by expressing the specific activity of the
[
C]glycogen as a percentage of the specific
activity of the [U-
C]glucose precursor. The
lower line is the hypothetical result if glycogenolysis specifically
removed the more recently incorporated
[
C]glycosyl residues prior to acting on the
older, preexisting glycogen. The horizontal line is the result expected
if glycogenolysis were completely random. The line through
experimentally derived values is the regression line excluding insulin
containing perfusions, since hearts synthesized glycogen in the
presence of insulin. The regression line had a slope of 0.403 g, dry
weight/µmol, and y intercept of 17.7%. r =
0.67, n = 15. Values are the mean ± S.E. for
four to six perfusions each. The symbols are: (
), perfusions
freeze-clamped at 45 min for determination of initial values; (
),
control (no addition at 45 min); (
), insulin (10 milliunits/ml)
added at 45 min; (
), glucagon (1 µg/ml) added at 45
min.
Table:
Balance
of total, [H]glycogen, and
[
C]glycogen
C]glycogen were calculated based on an initial
value of 89.3 ± 3.4 and 50.4 ± 7.5 µmol/g, dry
weight, respectably ( n = 6) determined in hearts
freeze-clamped at the beginning of the study period (45 min).
Table:
Rates of glycogenolysis, glycogen
synthesis, and glycolytic flux in perfused rat heart
H]glucose incorporation by correction for
simultaneous glycogenolysis. The derivation assumes that rates of
glycogen synthesis and degradation are constant, and that
[5-
H]glycosyl residues are representative of the
entire glycogen molecule with respect to susceptibility to
phosphorolysis. The instantaneous rate of incorporation of
[5-
H]glycosyl residues (net incorporation),
dN/dt, is the glycogen synthesis rate
( V
) minus the product of the rate of
glycogenolysis ( V
) and the fraction of the
glycogen occupied by [5-
H]glycosyl residues
( N/G
), where G
is the total glycogen content at time t:
is the initial glycogen content, then
in Equation 1 and
solving as a first order linear differential equation gives the
following:
= 1.50 µmol/min/g, dry weight,
t = 30 min, and N = 3.55 µmol/g,
dry weight ( i.e. the rate of net incorporation averaged over
30 min was 3.55/30 = 0.12 µmol/min/g, dry weight). The value
for G
(89.3 µmol/g, dry weight) was obtained
from perfusions that were terminated at the start of the study period
( i.e. freeze-clamped at 45 min). Solving for V
yielded 0.168 µmol/min/g, dry weight, for the rate of
glycogen synthesis.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.