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. 2013 Feb 1;288(5):2914-22.
doi: 10.1074/jbc.M112.409441. Epub 2012 Dec 12.

Evidence for transaldolase activity in the isolated heart supplied with [U-13C3]glycerol

Eunsook S Jin  1 , A Dean Sherry, Craig R Malloy
Affiliations

Evidence for transaldolase activity in the isolated heart supplied with [U-13C3]glycerol

Eunsook S Jin et al. J Biol Chem. .

Abstract

Studies of glycerol metabolism in the heart have largely emphasized its role in triglyceride synthesis. However, glycerol may also be oxidized in the citric acid cycle, and glycogen synthesis from glycerol has been reported in the nonmammalian myocardium. The intent of this study was to test the hypothesis that glycerol may be metabolized to glycogen in mammalian heart. Isolated rat hearts were supplied with a mixture of substrates including glucose, lactate, pyruvate, octanoate, [U-(13)C(3)]glycerol, and (2)H(2)O to probe various metabolic pathways including glycerol oxidation, glycolysis, the pentose phosphate pathway, and carbon sources of stored glycogen. NMR analysis confirmed that glycogen production from the level of the citric acid cycle did not occur and that the glycerol contribution to oxidation in the citric acid cycle was negligible in the presence of alternative substrates. Quite unexpectedly, (13)C from [U-(13)C(3)]glycerol appeared in glycogen in carbon positions 4-6 of glucosyl units but none in positions 1-3. The extent of [4,5,6-(13)C(3)]glucosyl unit enrichment in glycogen was enhanced by insulin but decreased by H(2)O(2). Given that triose phosphate isomerase is generally assumed to fully equilibrate carbon tracers in the triose pool, the marked (13)C asymmetry in glycogen can only be attributed to conversion of [U-(13)C(3)]glycerol to [U-(13)C(3)]dihydroxyacetone phosphate and [U-(13)C(3)]glyceraldehyde 3-phosphate followed by rearrangements in the nonoxidative branch of the pentose phosphate pathway involving transaldolase that places this (13)C-enriched 3-carbon unit only in the bottom half of hexose phosphate molecules contributing to glycogen.

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Figures

FIGURE 1.
FIGURE 1.
Possible metabolic routes for unequal 13C enrichment of carbons 1–3 versus 4–6 of glucosyl units of glycogen in heart supplied with [U-13C3]glycerol. A, during glucose production through standard gluconeogenic pathways, carbons 1–3 of glucose originate from DHAP whereas carbons 4–6 originate from GA3P. B, in hearts exposed to [U-13C3]glycerol, one would anticipate that incomplete equilibration at the level of TPI would yield more [1,2,3-13C3]glucosyl units than [4,5,6-13C3]glucosyl units because [U-13C3]glycerol is converted first to [U-13C3]DHAP before [U-13C3]GA3P is produced at the level of TPI. C, in comparison, rapid equilibration of [U-13C3]GA3P with S7P at the level of transaldolase would yield only [4,5,6-13C3]F6P and E4P. D, transaldolase exchange between carbons 4–6 of F6P and [U-13C3]GA3P would also yield only [4,5,6-13C3]F6P. OAA, oxaloacetate; E4P, erythrose 4-phosphate; CAC, citric acid cycle; open circles, 12C; filled circles, 13C. The x indicates that flow of 13C from the citric acid cycle into glycogen production is not considered in these examples. The asterisks in C and D indicate the carbons that are replaced by [U-13C3]GA3P through transaldolase activity to form [4,5,6-13C3]F6P.
FIGURE 2.
FIGURE 2.
13C NMR spectrum of MAG derived from hydrolyzed glycogen of a heart supplied with [U-13C3]glycerol. The multiplets seen in the C4, C5, and C6 resonances but not in the C1, C2, and C3 resonances indicate that carbons originating in [U-13C3]glycerol find their way only into the bottom half of glucosyl units of glycogen. The singlets detected in each glucosyl unit resonance reflect largely natural abundance levels of 13C. Q, doublet of doublets, or quartet, arising from coupling of C5 with both C4 and C6; S, singlet.
FIGURE 3.
FIGURE 3.
13C NMR spectrum of a heart tissue extract after perfusion with [U-13C3]glycerol. Only natural-abundance singlets were detected in glutamate, glutamine, and taurine, with the exception that [4,5-13C2]glutamate was observed at a very low level (0.022 ± 0.006%, n = 5). Resonance of glutamate C4 is expanded. The multiplets in lactate resonances are from [U-13C3]lactate, which is in exchange with [U-13C3]pyruvate in tissues. Because glutamate is in rapid exchange with α-ketoglutarate (α-kG), the presence of the small doublets due to 13C-13C spin-spin coupling between C4 and C5 (D45) indicates that a small portion of [U-13C3]pyruvate entered the citric acid cycle through acetyl-CoA. D45, doublet arising from coupling of C4 with C5; S, singlet.
FIGURE 4.
FIGURE 4.
Effect of isoproterenol pretreatment, insulin, or H2O2 on 13C enrichment and total glycogen content in hearts supplied with [U-13C3]glycerol. A and B, insulin increased the enrichment in the [4,5,6-13C3]glucosyl units of glycogen based on the 13C NMR analysis of hydrolyzed α-glucose C6 (A) and β-glucose C6 (B), but H2O2 decreased the enrichment. C, glycogen content remained the same by the presence of insulin or H2O2. Glycogen content was not different in hearts preperfused with isoproterenol compared with preperfusion with a substrate-free buffer without isoproterenol. glc C6, carbon 6 of glucosyl unit; #, p < 0.05; §, p < 0.01.
FIGURE 5.
FIGURE 5.
2H NMR spectrum of MAG derived from glycogen isolated from a heart supplied with [1,2-13C2]glucose and 2H2O. In the presence of 2H2O, deuterium (2H or D) enrichment is found in positions H1, H2, H4, and H5, but not in H3, H6R, and H6S. The absence of enrichment at H3 informs that DHAP did not contribute to carbons 1–3 of glucosyl units of glycogen through the standard glyconeogenic pathways (DHAP + GA3P → Fru-1,6-P2 → F6P → G6P → G1P → glycogen). The absence of 2H enrichment at the H6 position demonstrates the absence of glyconeogenesis from the citric acid cycle. The enrichment values shown in parentheses are average ± S.E. (n = 3). D1, D2, etc. indicate deuterium at glucosyl unit C1 position, C2 position, etc.
FIGURE 6.
FIGURE 6.
1H NMR and 13C NMR spectra from a heart supplied with [1,2-13C2]glucose and 2H2O. A, 1H NMR spectrum of hydrolyzed glycogen (H1 region of α-glucosyl units) from an isolated heart. The doublet due to scalar carbon-proton coupling in C1 of α-glucosyl units indicates that 23% of the glycogen was synthesized by direct phosphorylation of [1,2-13C2]glucose. B, glycolysis of [1,2-13C2]glucose produces [2,3-13C2]lactate. In contrast, flux through the oxidative arm of the PPP results in loss of carbon 1 with subsequent generation of [3-13C1]lactate. The singlet components in lactate C2 and C3 were essentially identical so must be assigned to natural abundance lactate. This shows that there was no significant flux of [1,2-13C2]glucose through the oxidative portion of the PPP. D23, doublet arising from coupling of C2 with C3; S, singlet due to natural abundance.
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