Contribution of Glycogen to Aerobic Myocardial Glucose Utilization
Background We determined glycogen turnover and the contribution of glycogen as a source of glucose for aerobic myocardial glycolysis and glucose oxidation in parallel series of isolated, working rat hearts subjected to pulse-chase perfusions.
Methods and Results Myocardial glycogen of isolated, working rat hearts was radiolabeled, after 30 minutes of substrate-free glycogen depletion, by perfusion for 60 minutes with buffer designed to stimulate resynthesis of glycogen (1.2 mmol/L palmitate, 11 mmol/L [U-14C]- or [5-3H]-glucose, 0.5 mmol/L lactate, and 100 μU/mL insulin). Rates of glucose oxidation (14CO2 production) and glycolysis (3H2O production) were then measured by perfusing the hearts for 40 minutes with buffer designed to simulate physiological conditions (0.4 mmol/L palmitate, 0.5 mmol/L lactate, 11 mmol/L [5-3H]- or [U-14C]-glucose, 100 μU/mL insulin) containing radiolabeled glucose different from that used during resynthesis. During the chase perfusion, rates of glycolysis and glucose oxidation from exogenous glucose were significantly greater than those from endogenous glycogen. Nevertheless, glycogen contributed significantly to myocardial energy production (41% of the overall ATP produced from glucose), and a significantly greater fraction of the glucose from glycogen that passed through glycolysis was oxidized (>50%) compared with the fraction oxidized from exogenous glucose (<20%, P<.05). Myocardial glycogen was simultaneously synthesized and degraded (ie, glycogen turnover) during the chase perfusion, despite net glycogenolysis. Furthermore, enrichment of labeled glucose in glycogen at the end of the chase perfusion, when corrected for newly synthesized glycogen, did not differ from that at the end of the labeling period.
Conclusions Thus, glycogen contributes significantly to aerobic myocardial glucose use under these experimental conditions, and the glucose derived from glycogen is oxidized preferentially compared with exogenous glucose. Additionally, substantial myocardial glycogen turnover occurs, and the manner in which glycogen is degraded does not fit the ordered hypothesis of “last glucose on, first glucose off.”
Normal myocardium uses a variety of energy substrates to meet its large energy requirements. Fatty acids, predominantly oleic and palmitic acid, are the preferred energy source of aerobically perfused normal hearts.1 2 Oxidation of fatty acids normally supplies 60% to 70% of the heart’s energy needs but may provide >90% of total ATP production under certain conditions.2 The heart also contains endogenous triacylglycerol stores that can serve as a source of fatty acids for oxidative metabolism2 and can provide from 11% to 50% of the heart’s energy requirements (depending on extracellular fatty acid supply).2
Exogenous glucose and lactate are the other major energy substrates of the myocardium.1 2 Glucose is metabolized by glycolysis and mitochondrial oxidation,1 2 whereas lactate may be oxidized as well as produced by the myocardium.3 As with fatty acids, exogenous glucose can also be incorporated into an endogenous storage form, glycogen.1 Since its discovery, detailed study and characterization of glycogen metabolism have led to the development of many important concepts of enzyme regulation and mechanisms of hormone action.4 5 Despite this extensive research activity, a number of key issues regarding myocardial glycogen metabolism remain to be resolved.
One key question is what fraction of glucose taken up by the myocardium directly enters glycolysis and what fraction is converted into its storage form, glycogen. Studies are divided as to the extent to which glucose taken up by the heart is converted into glycogen. For example, studies in humans have suggested that only 20% of the glucose extracted by the myocardium is immediately oxidized and only 13% is metabolized to lactate.6 Although not measured directly, it was estimated that 60% to 70% of the glucose taken up by the heart was incorporated into glycogen.6 However, direct measurements suggest that only 4.5% to 7.5% of glucose extracted by isolated, working rat hearts is used to synthesize glycogen.7 The explanation for the discrepancy between these two studies is not immediately apparent. Although differences in species and preparation may contribute to this discrepancy, the fact that the isolated rat hearts were perfused with buffer containing carbohydrate but no fatty acid may be an additional factor.7 It is well known that fatty acids significantly affect myocardial glucose and glycogen metabolism by reducing glucose use and favoring glycogen synthesis.1 8
A second unresolved issue is whether exogenous glucose and glucose from glycogen share a similar fate in the heart. When used by the myocardium, glucose from glycogen generally has been believed to be metabolized like its exogenous counterpart. Investigations of glucose use by vascular smooth muscle suggest that significant differences exist with respect to the fates of exogenous glucose and glucose from glycogen.9 10 In studies reported by Lynch and Paul,9 it was found that glycogen use correlated with oxidative phosphorylation and contractile activity, whereas glucose uptake and aerobic glycolysis were associated with release of lactate, suggesting that exogenous and endogenous glucose were metabolized by separate pathways. Since direct measurements of the contribution of glycogen to both glycolytic and oxidative pathways have not been performed in the heart, it is not known whether myocardial utilization of glucose from glycogen differs from that of exogenous glucose.
A third unresolved issue is whether glycogen is actively “turning over” in the heart under normal circumstances. The pathways of glycogen synthesis and glycogenolysis are separate,1 involving different enzymes that are regulated in a reciprocal manner such that glycogenolysis is inhibited when synthesis is stimulated and vice versa.1 The completeness of this reciprocal regulation and the extent to which simultaneous synthesis and degradation of glycogen (ie, glycogen turnover) occurs in aerobic myocardium, however, remain to be fully characterized. As occurs with endogenous triacylglycerol stores,2 Goodwin et al7 demonstrated that significant glycogen turnover occurs in aerobic, isolated, working rat hearts perfused in the absence of insulin and fatty acids. On the other hand, no significant glycogen turnover was detectable in hearts of insulin-treated rats studied in vivo by nuclear magnetic resonance (NMR) imaging11 or in isolated, working rat hearts perfused with buffer containing pharmacological concentrations of insulin but no fatty acids.7 Whether glycogen turnover occurs in the presence of fatty acids and more physiological concentrations of insulin remains to be determined.
Finally, it is also not clear whether glycogen is synthesized and degraded in an ordered or a random manner. Glycogen is a very large molecule with a branched structure that exists in multiple tiers.12 13 Brainard et al14 used [13C]-glucose and NMR methodology to study glycogen metabolism in isolated, Langendorff-perfused guinea pig hearts. They concluded that glycogen synthesis and mobilization was “mostly” ordered (ie, first glucose molecule added to glycogen was the last to be mobilized). By this reasoning, the last glucose molecule added to glycogen should also be the first off. However, Goodwin et al7 have recently provided data from isolated, perfused rat hearts that do not support the ordered concept and suggest that glycogenolysis is substantially random.
The present study was performed to address the above controversies in myocardial glycogen metabolism. The overall objectives were (1) to determine the contribution of glycogen to aerobic glucose use; (2) to determine whether glucose from glycogen is oxidized preferentially, compared with exogenous glucose; (3) to measure the extent of glycogen turnover; and (4) to assess the degree of orderedness of glycogen degradation in the heart. To accomplish these objectives, we studied parallel series of pulse-chase perfusions in isolated, fatty acid–perfused, working rat hearts in which glycogen was labeled with either [3H]- or [14C]-glucose and that were subsequently perfused with buffer containing [3H]- or [14C]-glucose different from that present in glycogen.
Bovine albumin (fraction V) was obtained from Boehringer-Mannheim Canada, beef-pork insulin was obtained from Eli Lilly, and radiochemicals were obtained from NEN-Du Pont. Dowex 1-X4 anion exchange resin (200 to 400 mesh) was obtained from Bio-Rad. All other chemicals were obtained from Sigma Chemical Co or BDH and were of analytical grade.
Isolated Heart Preparation
Hearts from fed, male Sprague-Dawley rats (weight, 350 to 450 g) anesthetized with halothane (2% to 3%) were excised and placed in ice-cold buffer, and the aorta was quickly cannulated.15 16 Hearts were initially perfused via the aorta in the Langendorff mode with oxygenated Krebs-Henseleit buffer, pH 7.4, containing 11 mmol/L glucose and 2.5 mmol/L calcium, at a constant pressure of 60 mm Hg. During the initial 10-minute retrograde aortic perfusion, hearts were trimmed of excess tissue and the left atrium was cannulated. The hearts were switched to the working mode after an additional 30 minutes of aerobic, substrate-free, retrograde aortic perfusion (see below) and were perfused with recirculating buffer at a left atrial preload of 11.5 mm Hg and an aortic afterload of 80 mm Hg. During the working mode, heart rate and peak systolic pressure were recorded by use of a DIREC physiological recording system (Fine Science Tools Inc) with a pressure transducer (Viggo-Spectramed) in the aortic afterload line.
Parallel series of pulse-chase perfusions (Fig 1⇓) were used to measure myocardial glucose use and glycogen metabolism in the isolated, perfused hearts. Myocardial glycogen was initially depleted by a 30-minute Langendorff perfusion with oxygenated substrate-free, insulin-free buffer. Hearts were then switched to the working mode and glycogen was resynthesized during a 60-minute pulse glycogen-labeling period with either [U-14C]-glucose (series A, 29 104±2063 disintegrations per minute per micromole (dpm/μmol) of glucose, n=6) or [5-3H]-glucose (series B, 32 740±299 dpm/μmol glucose, n=7). During this period, buffer contained 1.2 mmol/L palmitate, 11 mmol/L [5-3H]- or [U-14C]-glucose, 0.5 mmol/L lactate, and 100 μU/mL insulin. Insulin and high fatty acid concentrations were included to stimulate glycogen synthesis. After 60 minutes, hearts were switched to a recirculating perfusion buffer circuit that allowed determination of both exogenous and endogenous glucose utilization during the subsequent 40-minute chase perfusion. During this perfusion period, hearts were perfused with buffer containing 0.4 mmol/L palmitate, 11 mmol/L [5-3H]- or [U-14C]-glucose, 0.5 mmol/L lactate, and 100 μU/mL insulin. The [5-3H]-glucose (series A, 34 821±1951 dpm/μmol glucose, n=6) or [U-14C]-glucose (series B, 23 944±1910 dpm/μmol glucose, n=7) used during the chase period was opposite to the radiolabel used during glycogen labeling.
Rates of glucose oxidation and glycolysis from [14C]- or [3H]-labeled glycogen and [U-14C]- or [5-3H]-glucose were measured simultaneously during the chase perfusion by quantitatively measuring rates of 14CO2 production (oxidation) and 3H2O production (glycolysis), respectively. In one series (series A, Fig 1⇑), we determined the rate of glucose oxidation (14CO2 production) from endogenous glycogen during the chase period at the same time as rates of glycolysis (3H2O production) from exogenous glucose were measured. In the second series (series B, Fig 1⇑), we measured the rate of glycolysis from endogenous glycogen during the chase period while simultaneously measuring rates of glucose oxidation from exogenous glucose. In this manner, the contribution of endogenous glycogen and exogenous glucose to both glucose oxidation and glycolysis was determined. Approximately 5% to 10% of labeled glucose used during the preceding glycogen labeling period was carried over into the chase perfusate. The degree of contamination was determined by measuring specific activity of the buffer at the beginning of the chase perfusion. Correction for the contribution of this exogenous source of glucose to rates of glycolysis or oxidation measured from glycogen was made using the appropriate mean rate of glycolysis or glucose oxidation from exogenous glucose during the chase period of the opposite series.
A series of hearts were quick-frozen at various time points throughout the protocol by clamping with aluminum tongs cooled to the temperature of liquid nitrogen. This included hearts frozen immediately after removal from anesthetized rats, at the end of the glycogen depletion period, at the end of the glycogen labeling period, and at the end of the chase perfusion. Relative rates of glycogen synthesis and glycogenolysis during the chase period were determined by comparing myocardial glycogen content and specific activity in hearts that were frozen either at the end of the pulse perfusion or at the end of the chase perfusion.
Measurement of Glucose Oxidation and Glycolysis
Steady state rates of oxidation of glucose were determined by measurement of the rate of 14CO2 production, as described previously.15 16 17 During the chase perfusion, hearts were perfused in a closed system that allowed quantitative collection of gaseous and perfusate 14CO2 originating from either exogenous 14C-glucose (series B) or endogenous 14C-glucose (series A). Perfusate and gaseous samples were collected at 2, 5, and 10 minutes and then at 10-minute intervals throughout the 40-minute chase period. The 14CO2 liberated in the gaseous state was trapped in a 1 mol/L hyamine hydroxide solution in the gas outlet line. Samples of this solution were injected directly into scintillation liquid for counting. Perfusate samples were immediately injected below a 1 mL volume of mineral oil to prevent liberation of perfusate 14CO2. The 14CO2 from the perfusate was subsequently extracted by injection of 1 mL of perfusate into a sealed, metabolic flask containing 9N H2SO4 and 400 μL of 1 mol/L hyamine hydroxide in a suspended center well. The flasks were gently shaken for 1 hour to release the 14CO2 present as [14C]-bicarbonate. The center wells were then removed and counted in scintillation liquid by use of standard counting procedures. Glucose oxidation rates were expressed as nanomoles of glucose oxidized per minute per gram of dry heart weight (dry wt).
Quantitative 3H2O production was used to measure steady state glycolytic rates. 3H2O is liberated from [5-3H]-glucose at the triose phosphate isomerase and enolase steps of glycolysis. Samples for 3H2O originating from either exogenous 3H-glucose (series A) or endogenous 3H-glucose (series B) were collected during the chase period at the same time intervals as the 14CO2. 3H2O was separated from [3H]-glucose and [14C]-glucose by use of columns containing Dowex 1-X 4 anion exchange resin (200 to 400 mesh) suspended in 0.4 mol/L potassium tetraborate.2 15 The Dowex in the columns was extensively washed with H2O before use. A 0.2 mL volume of perfusate was added to the column and eluted into scintillation vials with 0.8 mL H2O. The samples were then subjected to standard double-isotope counting procedures. The Dowex columns were found to retain 98% to 99.6% of the total [3H]-glucose and [14C]-glucose present in the perfusate. The 3H2O was corrected for the small proportion of [3H]-glucose that passed through the column. This could be accomplished because an equal amount of [14C]-glucose also passed through the column and could be used as an internal standard for the degree of [3H]-glucose contamination in the 3H2O sample. Correction was also made for the degree of spillover of [14C] into the [3H] counting window by measuring this degree of spillover with standards containing [14C]-glucose. Glycolytic rates were expressed as nanomoles of glucose metabolized per minute per gram of dry heart weight.
The frozen ventricular tissue was weighed and powdered in a mortar and pestle cooled to the temperature of liquid nitrogen. A portion of the tissue was then used to determine the ratio of dry to wet weight. Myocardial glycogen was determined in the powdered ventricular tissue as glucose after digestion with 30% KOH, ethanol precipitation, and acid hydrolysis of glycogen.18 A portion of the glycogen extract was used for scintillation counting to determine enrichment and specific activity of the glycogen pool by [3H] and [14C].
Data are expressed as mean±SEM. Heart function was analyzed by repeated measures ANOVA with one grouping factor and one repeating factor. Rates of ATP production and initial and final labeled glycogen were analyzed by two-way ANOVA. Rates of glycolysis and glucose oxidation were compared by two-way ANOVA after log transformation. Enzymatically determined glycogen was analyzed by one-way ANOVA, whereas glycogen enrichment was compared by independent Student’s t tests. Ratios of glucose oxidation and glycolysis were calculated as described by Mood et al19 and compared by examining 95% CIs. A sequential rejective Bonferroni procedure was applied to all tests to correct for multiple tests and/or multiple comparisons. A corrected value of P<.05 was considered significant.
Heart Function During Glycogen Labeling and Chase Perfusions
A number of initial experiments were performed in an attempt to optimize conditions for glycogen depletion in the heart without compromising heart function during the chase. The protocols included the use of hypoxic perfusion, inclusion of glucagon in aerobic and hypoxic perfusate, and substrate-free perfusion of the aerobic working heart with and without electric pacing. Although many of these protocols successfully depleted myocardial glycogen stores, heart function almost always was also compromised. A protocol found to preserve heart function while substantially depleting glycogen involved a 30-minute Langendorff perfusion in the absence of substrate and insulin. Table 1⇓ summarizes the heart rate, peak systolic pressure, and heart rate multiplied by peak systolic pressure product data in working hearts perfused during pulse and chase perfusions. No significant differences were observed between groups at the end of either the pulse-labeling or chase periods. Left ventricular function, calculated as either the heart rate multiplied by peak systolic pressure product or peak systolic pressure, showed a small but significant decline during the chase period. Heart rate at the end of the pulse period was not different from that at the end of the chase perfusion in either group.
The profile of changes that occurred in the glycogen storage pool during the course of the experiments is illustrated in Fig 2⇓. Each group represents a series of hearts frozen at selected time points throughout the protocol. As can be seen from Fig 2⇓, myocardial glycogen was significantly depleted by the 30-minute period of aerobic substrate-free perfusion to 39.5±9.1% of baseline values. During the subsequent glycogen labeling period, a substantial resynthesis of glycogen occurred, with levels of myocardial glycogen returning to baseline values. During the chase-perfusion period, when glucose utilization from exogenous glucose and endogenous glycogen was measured, a small but significant decrease in net glycogen levels occurred, with myocardial glycogen dropping to 70.7±17.6% of values at the end of pulse labeling for an overall loss of 32.4±4.3 μmol/g dry wt glycogen.
Rates of Glycolysis and Glucose Oxidation
During the chase period, accumulation of 3H2O from glycolysis and 14CO2 from oxidation of glucose moieties was linear (data not shown) over time after the first 5 to 10 minutes. Mean rates of glycolysis and glucose oxidation from exogenous glucose and endogenous glycogen, calculated from rates at 20, 30, and 40 minutes of perfusion, are summarized in Fig 3⇓. Rates of glycolysis from exogenous glucose (2768±72 nmol · min−1 · g−1 dry wt) were significantly greater than those from endogenous glycogen (792±26 nmol · min−1 · g−1 dry wt, P<.05). Rates of glucose oxidation from exogenous sources (526±8 nmol · min−1 · g−1 dry wt) were also significantly greater than oxidation of glucose from glycogen (416±3 nmol · min−1 · g−1 dry wt, P<.05).
As has been well described previously,2 only a portion of the exogenous glucose that passed through glycolysis was oxidized (Fig 3⇑). The same general pattern held true for rates of glycolysis and oxidation of glucose that originated from glycogen (Fig 3⇑). However, the fraction of glucose from glycogen that passed through glycolysis and was subsequently oxidized (0.53±0.14) was significantly greater than the fraction of exogenous glucose passing through glycolysis that was oxidized (0.19±0.01, P<.05). In other words, >50% of glucose from glycogen was oxidized compared with <20% of exogenous glucose. The majority of 14C in myocardial extracts was found to be present in the glycogen fraction, and calculations based on contents of myocardial glycolytic and tricarboxylic acid cycle intermediates reported in the literature20 indicate that nonglycogen sources are not likely to account for the preferential oxidation of glycogen observed (data not shown).
Rates of ATP Production From Myocardial Glucose Utilization
Rates of ATP produced by myocardial glucose utilization were calculated from mean rates of glycolysis and glucose oxidation assuming 2 moles ATP were produced for each mole of exogenous glucose passing through glycolysis, 3 moles of ATP for each mole of glucose from glycogen passing through glycolysis, and 36 moles of ATP for each mole of exogenous and endogenous glucose oxidized to CO2. The data, summarized in Fig 4⇓, show that metabolism of glycogen by glycolysis and oxidation contributes significantly to ATP production from myocardial glucose use. In fact, 41% of ATP produced from glucose metabolism by these hearts under these experimental conditions is derived from glycogen. The vast majority of ATP from both sources is derived from oxidation of glucose.
Myocardial Glycogen Balance
Table 2⇓ summarizes the changes observed in the content of [3H]-glucose and [14C]-glucose in myocardial glycogen during the chase period. The results of series A were not significantly different from those of series B. Substantial quantities of labeled glucose were incorporated into glycogen during the labeling period such that 45% to 46% of the glycogen pool was labeled. On the basis of changes in activity in myocardial glycogen between hearts at the end of the labeling period and those at the end of the chase perfusion and calculations made with the specific activity of glucose in the labeling buffer, 23.8±5.0 μmol/g dry wt of [3H]-glycogen and 22.8±5.3 μmol/g dry wt of [14C]-glycogen were released during the 40-minute chase period. Interestingly, despite this substantial degree of glycogenolysis, net glycogen synthesis occurred simultaneously, as indicated by incorporation of [14C]-glucose and [3H]-glucose into glycogen during the chase period. Accordingly, one would have expected a net loss of glycogen ranging from 3.8±6.4 μmol/g dry wt for [3H]-glycogen to 10.3±8.2 μmol/g dry wt for [14C]-glycogen. However, myocardial glycogen content measured enzymatically in these same hearts was reduced by 29.5±6.0 mmol/g dry wt for [3H]-glycogen and 36.0±6.4 mmol/g dry wt for [14C]-glycogen during the chase perfusion. This suggests that in addition to turnover of radiolabeled glycogen, a significant amount of unlabelled glucose has also been released from glycogen during this period.
If the calculations are repeated assuming that degradation of glycogen is random, then total glycogen loss may be determined as the quotient of the change in labeled glycogen (23.8±5.0 μmol/g dry wt for [3H]-glycogen and 22.8±5.3 μmol/g dry wt for [14C]-glycogen) and the enrichment of glycogen (ie, the specific activity averaged over all glycogen at the end of the pulse perfusion divided by the specific activity of labeling buffer; 0.44±0.05 for [3H]-glycogen and 0.48±0.02 for [14C]-glycogen). Accordingly, it is anticipated that a reduction of glycogen content ranging from 46.1±10.7 to 51.0±10.2 μmol/g dry wt should occur during the chase period, assuming no simultaneous synthesis. If simultaneous incorporation of labeled glucose is taken into account by calculating the amount of glycogen synthesized during the chase (determined from the activity of [3H]- or [14C]-glucose in glycogen and the corresponding specific activity of the chase buffer), the predicted change in glycogen ranges from 31.0±10.1 μmol/g dry wt for [3H]-glycogen to 34.3±14.4 μmol/g dry wt for [14C]-glycogen. These values agree well with the measured changes in glycogen content.
Glycogen is a storage form of glucose that was formerly believed to make insignificant contributions to energy metabolism of the myocardium under aerobic conditions.1 Glycogen use is thought to increase if hearts are exposed to increased workloads,21 perfused without insulin and/or fatty acid,7 21 or subjected to hypoxic/ischemic conditions.1 As shown in the present study, net glycogenolysis occurred and substantial quantities of glucose from glycogen were metabolized by glycolysis and glucose oxidation in working hearts despite the presence of insulin and physiological levels of fatty acid. Our data also demonstrate that the contribution of glycogen as a source of glucose has been underestimated in previous studies because simultaneous glycogenolysis and glycogen synthesis was not considered. Although rates of glycolysis and oxidation of glucose from glycogen were less than rates of glycolysis and oxidation of exogenous glucose (Fig 3⇑), glycogen metabolism contributed significantly to overall ATP production. In fact, glucose from glycogen accounted for 41% of the ATP produced from myocardial glucose utilization (Fig 4⇑).
The vast majority of myocardial ATP was produced by mitochondrial oxidation of both exogenous and endogenous glucose (Fig 4⇑). By combining data from the present study with substrate utilization data from other studies of isolated, working rat hearts perfused under similar conditions,2 15 we estimated the contribution of glycogen to aerobic energy metabolism. Under these conditions, exogenous palmitate is the preferred energy substrate (>50% of ATP produced), whereas glucose and lactate oxidation contribute ≈14% and 5% of the ATP produced, respectively. Utilization of endogenous fatty acid (triacylglycerol) and glucose (glycogen) produces ≈25% of the ATP, with glycogen accounting for about 12% to 13%. Overall, glycolysis produces 7% of the total ATP, with ≈1% to 2% coming from glycolysis of glucose from glycogen. Since the rates were not corrected for isotopic dilution of glycogen, the rate of ATP production from glycogen represents a minimal estimate.
Until the present study, it was assumed but not confirmed by experimental evidence that glucose from myocardial glycogen is metabolized in the same manner as exogenous glucose. Our data indicate that, in fact, glucose from glycogen is preferentially oxidized compared with exogenous glucose (Fig 3⇑). More than 50% of glucose from glycogen passing through glycolysis is oxidized, whereas <20% of exogenous glucose passing through glycolysis is oxidized, a statistically significant difference (P<.05). This finding is entirely in keeping with observations in vascular smooth muscle.9 10 In the myocardium, De Tata et al22 presented evidence for transmural gradients of glycogen metabolism. Our data suggest that energy metabolism is also compartmentalized within cardiac myocytes, a view consistent with reports by others23 24 25 26 that proposed that carbohydrate metabolism in the myocardium is regionalized. The significance of this preferential oxidation and whether glycogenolysis is coordinated with oxidative phosphorylation and mechanical activity remain to be determined.
Despite the presence of insulin and physiological levels of fatty acid (0.4 mmol/L palmitate) in the experiments in the current study, net myocardial glycogen loss occurred during the chase perfusion (Fig 2⇑). Saddik and Lopaschuk2 found that the myocardial triacylglycerol pool also decreased in size in the presence of physiological levels of fatty acid. They speculated that 0.4 mmol/L palmitate was not the concentration of fatty acid in the interstitial compartment seen by the heart in vivo and, therefore, endogenous pools of triacylglycerol were reduced. The same may hold true for glycogen metabolism. In the face of subphysiological perfusate fatty acid levels, glycogen was used to generate energy.
We also found that significant turnover of glycogen occurred during the aerobic chase period in the presence of substantial glycogenolysis and an overall net reduction of myocardial glycogen content, in keeping with the work of Goodwin et al.7 Whether myocardial glycogen turnover occurs when net glycogen content does not change remains to be determined. Rates of net synthesis, calculated from the net incorporation of glucose into glycogen during the chase period, ranged from 0.33 to 0.51 μmol · min−1 · g−1 dry wt. Although these rates of synthesis are likely an underestimate, since they are not corrected for simultaneous glycogenolysis of newly synthesized glycogen, they do compare favorably with those obtained by Goodwin et al7 (0.17 to 0.62 μmol · min−1 · g−1 dry wt). Glucose taken up by the heart is either metabolized or synthesized into a storage form.1 27 Under the experimental conditions used in the present study, we found that ≈11.6% of the glucose taken up by the heart was synthesized into glycogen. This value compares favorably with that observed by Goodwin et al,7 who found that 4.5% to 7.5% of glucose taken up by the heart is synthesized into glycogen. On the other hand, our data are inconsistent with those of Wisneski et al6 and do not support the concept that the majority of glucose taken up by the heart cycles through glycogen, as suggested by these investigators on the basis of indirect measurements in humans. The fact that the value in the current study is higher than that observed by Goodwin et al7 is presumably a consequence of fatty acid in the buffer, a situation that would favor glycogen synthesis. The reason for the dramatic difference between the values in the current study and those of Wisneski et al6 is not immediately clear but may be related to methodological differences.
Glycogen is a very large molecule with a branched structure that exists in multiple tiers.12 13 Whether glycogen is synthesized and degraded in an ordered or a random manner remains controversial.7 14 When changes in myocardial glycogen content and specific activity were considered and newly incorporated glucose was taken into account, we found the best agreement between measured changes in glycogen content and loss of labeled glucose moieties occurred when random degradation was assumed. On the basis of this assumption, estimated loss of glycogen ranged from 31.0±10.1 to 34.3±14.4 μmol/g dry wt during the chase period, values that agree well with the overall measured loss of glycogen of 32.8±4.3 μmol/g dry wt. Furthermore, calculation of glycogen enrichment at the end of the chase period ([3H]-glycogen, 0.45±0.08 dpm/μmol, n=6; [14C]-glycogen 0.47±0.14 dpm/μmol, n=6), with the newly incorporated glucose taken into account, demonstrates that glycogen enrichment, averaged over all the glycogen, does not change significantly in comparison with that at the end of the labeling period ([3H]-glycogen, 0.44±0.05 dpm/μmol, n=5; [14C]-glycogen, 0.48±0.02 dpm/μmol, n=4, P=NS). These findings are in keeping with those of Goodwin et al7 and are consistent with the interpretation that glycogen is degraded in a substantially random manner. Although we anticipated that measured rates of glycolysis from endogenous sources would correspond to loss of labeled glucose from myocardial glycogen, rates of glycolysis were ≈25% to 30% higher than expected on the basis of the loss of labeled glucose from glycogen. The explanation for this lack of correspondence is unclear.
On the basis of these experiments, several important and novel conclusions may be made regarding aerobic myocardial glycogen metabolism. In aerobic, isolated, working rat hearts perfused with insulin and physiological concentrations of fatty acid, net glycogenolysis occurs and glycogen contributes significantly to overall myocardial glucose utilization, accounting for 41% of the ATP produced. An additional observation arising from these studies is that glucose from myocardial glycogen is oxidized preferentially compared with exogenous glucose, consistent with the concept that myocardial carbohydrate metabolism is compartmentalized. Furthermore, substantial glycogen turnover (ie, simultaneous synthesis and degradation) occurs in the aerobic heart in the presence of net glycogenolysis. Under the conditions of study, at least 11.6% of glucose taken up by the heart is directed toward glycogen synthesis despite net glycogenolysis. Finally, our data support the concept that myocardial glycogen is degraded in a random as opposed to an ordered manner.
Note Added in Proof
Since this paper was accepted for publication, Goodwin et al have also presented data supporting the concept that glucose from glycogen is preferentially oxidized in the heart (Circulation. 1995;92:I-769. Abstract.).
This study was supported by grants from the Medical Research Council of Canada and the Heart and Stroke Foundation of B.C. & Yukon. Dr Allard is a Research Scholar of the Heart & Stroke Foundation of Canada. Dr Lopaschuk is a Medical Research Council of Canada Scientist and an Alberta Heritage Foundation for Medical Research Senior Scholar. S.L. Henning and B.O. Schönekess are graduate student trainees of the Heart and Stroke Foundations of B.C. & Yukon (S.L.H.) and Canada (B.O.S.). B.O. Schönekess is also a graduate student trainee of the Alberta Heritage Foundation for Medical Research. The authors thank Lorraine Verburgt for help with statistical analyses and Stuart Greene for assistance with the figures.
Reprint requests to Michael F. Allard, BSc, MD, FRCPC, Department of Pathology and Laboratory Medicine, University of British Columbia, Cardiovascular Research Laboratory, St Paul’s Hospital, 1081 Burrard St, Vancouver, BC, Canada V6Z 1Y6. E-mail email@example.com.
- Received August 1, 1995.
- Revision received October 23, 1995.
- Accepted November 5, 1995.
- Copyright © 1996 by American Heart Association
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