©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Glycogen Turnover in the Isolated Working Rat Heart (*)

Gary W. Goodwin , James R. Arteaga , Heinrich Taegtmeyer (§)

From the (1) University of Texas Houston Medical School, Division of Cardiology, Department of Internal Medicine, Houston, Texas 77030

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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-C]glucose, glycogenolysis was determined continuously from the washout of COplus [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 (HO from [5-H]glucose) as well as total glycolytic flux (HO 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.


INTRODUCTION

The experimental reports regarding simultaneous glycogen synthesis and degradation, mostly pertaining to liver, are conflicting. Glycogen turnover is indicated in the liver by 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.

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-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 HO, 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.

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.


EXPERIMENTAL PROCEDURES

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 COas 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 CaClconcentration 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 HO.

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-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 HO (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, HO, 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 COand includes COaccumulating in the perfusate as well as COexhausted 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 COtrap contained within a scintillation vial with the use of a gas dispersion tube. The exhausted COwas 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 COin the perfusate was determined by trapping COliberated 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 COwas 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 COfor 5 min before cannulating the heart and by trapping COfrom 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 COwas 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 HO 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.

Hearts, stored at -70 °C, were weighed, powdered under liquid N, 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.


RESULTS

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-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).

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 [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 COplus [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 COplus [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.

Fig. 2 shows the time course of release of COplus [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 COplus [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 COplus [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 COplus [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.

Glycolytic fluxes are presented in , and include flux from exogenous glucose (HO 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 HO. Glycogen breakdown () was determined from the release of COplus [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.




DISCUSSION

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-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).

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 [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.

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 COplus [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 COplus [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 COplus [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 ().

The reasonably good agreement between measured and predicted glycogen balances illustrated above supports the assumption of uniform isotopic dilution of [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) .

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 [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

Values are the mean ± S.E. in µmol/g, dry weight. Changes in total and [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



FOOTNOTES

*
This work was supported by United States Public Health Service Grant RO1 HL-43133. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: University of Texas Houston Medical School, Division of Cardiology, Dept. of Internal Medicine, 6431 Fannin, Houston, TX 77030. Tel.: 713-794-4125; Fax: 713-792-5187.


ACKNOWLEDGEMENTS

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-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 Gis the total glycogen content at time t:

On-line formulae not verified for accuracy

If Gis the initial glycogen content, then

On-line formulae not verified for accuracy

Substituting for Gin Equation 1 and solving as a first order linear differential equation gives the following:

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= 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 Vyielded 0.168 µmol/min/g, dry weight, for the rate of glycogen synthesis.


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