Interaction between the pentose phosphate pathway and gluconeogenesis from glycerol in the liver

Abstract

After exposure to [U-(13)C3]glycerol, the liver produces primarily [1,2,3-(13)C3]- and [4,5,6-(13)C3]glucose in equal proportions through gluconeogenesis from the level of trioses. Other (13)C-labeling patterns occur as a consequence of alternative pathways for glucose production. The pentose phosphate pathway (PPP), metabolism in the citric acid cycle, incomplete equilibration by triose phosphate isomerase, or the transaldolase reaction all interact to produce complex (13)C-labeling patterns in exported glucose. Here, we investigated (13)C labeling in plasma glucose in rats given [U-(13)C3]glycerol under various nutritional conditions. Blood was drawn at multiple time points to extract glucose for NMR analysis. Because the transaldolase reaction and incomplete equilibrium by triose phosphate isomerase cannot break a (13)C-(13)C bond within the trioses contributing to glucose, the appearance of [1,2-(13)C2]-, [2,3-(13)C2]-, [5,6-(13)C2]-, and [4,5-(13)C2]glucose provides direct evidence for metabolism of glycerol in the citric acid cycle or the PPP but not an influence of either triose phosphate isomerase or the transaldolase reaction. In all animals, [1,2-(13)C2]glucose/[2,3-(13)C2]glucose was significantly greater than [5,6-(13)C2]glucose/[4,5-(13)C2]glucose, a relationship that can only arise from gluconeogenesis followed by passage of substrates through the PPP. In summary, the hepatic PPP in vivo can be detected by (13)C distribution in blood glucose after [U-(13)C3]glycerol administration.

Keywords: Gluconeogenesis; Glucose Metabolism; Glycerol; Liver Metabolism; Nuclear Magnetic Resonance (NMR); Pentose Phosphate Pathway (PPP).

© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Figures

FIGURE 1.

13C symmetry between the top and bottom half of glucose from liver supplied with [U-13C3]glycerol. DHAP and GA3P condense during gluconeogenesis. Carbons 1–3 of glucose originate from DHAP, whereas carbons 4–6 originate from GA3P (A). The liver would produce equal amounts of [1,2,3-13C3]glucose and [4,5,6-13C3]glucose from [U-13C3]glycerol if there is complete equilibrium at the level of TPI, no PPP, and no transaldolase activity (B). If a small portion of [U-13C3]glycerol enters the CAC prior to gluconeogenesis, a set of GA3P isotopomers ([2,3-13C2]-, [1,2-13C2]-, [U-13C3]GA3P) are produced, which are common intermediates for both top and bottom half carbons of glucose. Consequently, there would be equal in the ratios of [1,2-13C2]/[2,3-13C3] and [5,6-13C2]/[4,5-13C2] if the PPP is not active (C). Transaldolase activity or disequilibrium at the level of TPI does not influence the relative amount of [1,2-13C2]- and [2,3-13C2]glucose (or the relative amount of [5,6-13C2]- and [4,5-13C2]glucose) because neither pathway disrupts the information encoded by the 13C distribution within the three-carbon unit. OAA, oxaloacetate; open circle, 12C; filled circle, 13C.

FIGURE 2.

13C Asymmetry between the top and bottom half of glucose from the liver supplied with [U-13C3]glycerol. Any lack of complete equilibrium at the level of TPI causes [1,2,3-13C3]glucose > [4,5,6-13C3]glucose because [U-13C3]glycerol was converted to [U-13C3]DHAP first before [U-13C3]GA3P through TPI (A). After [U-13C3]glycerol phosphorylation, [U-13C3]GA3P was produced through TPI activity. Transaldolase reaction between [U-13C3]GA3P and sedoheptulose 7-phosphate (S7P) produces [4,5,6-13C3]F6P and erythrose 4-phosphate, and consequently [4,5,6-13C3]glucose (B). After [U-13C3]glycerol administration, [1,2,3-13C3]- and [4,5,6-13C3]hexose are the main isotopomers through gluconeogenesis from the level of trioses. The entry of [1,2,3-13C3]G6P into the PPP produces primarily [1,2-13C2]F6P, and consequently [1,2-13C2]glucose, but the PPP does not change the labeling patterns in bottom half carbons of the hexose (C). The entry of [U-13C3]glycerol into the CAC prior to gluconeogenesis would result in [1,2-13C2]/[2,3-13C3] = [5,6-13C2]/[4,5-13C2] in glucose if the PPP is not active, but [1,2-13C2]/[2,3-13C2] ≥ [5,6-13C2]/[4,5-13C2] if the PPP is active. Thus, the difference of [1,2-13C2]/[2,3-13C2] and [5,6-13C2]/[4,5-13C2] in glucose is sensitive to the hepatic PPP activity. E4P, erythrose 4-phosphate; GNG, gluconeogenesis.

FIGURE 3.

[1,2-13C2]F6P Formation from [1,2,3-13C3]G6P through the pentose phosphate pathway. The carbon 1 of G6P is decarboxylated in the oxidative phase of the PPP producing Ru5P; these carbons are further rearranged in the non-oxidative phase. The entry of three G6P molecules into the pentose pathway produces two F6P molecules and one GA3P molecule. If [1,2,3-13C3]G6P enters the pathway with unlabeled two G6P molecules, [1,2-13C2]F6P is formed through the routes illustrated in A or B. [3-13C1]F6P can be also produced through alternative pathways (C). The singly labeled hexose is not considered in this study to avoid complexity associated with natural abundance. Note that the order in carbons 4–6 of G6P remains the same through the PPP. All carbon numbers in this figure indicate the original carbon positions of G6P before the entry into the PPP. Metabolites underlined (F6P and GA3P) are the products after the PPP. Ru5P, ribulose 5-phosphate; R5P, ribose 5-phosphate; Xu5P, xylulose 5-phosphate; open circle, 12C; gray circle, 13C.

FIGURE 4.

Assessment of hepatic PPP activity based on NMR analysis of glucose using [U-13C3]glycerol versus [U-13C3]lactate. Rats received a mixture of glucose, [U-13C3]glycerol, and lactate or a mixture of glucose, glycerol, and [U-13C3]lactate after a fast or without a fast. A shows 13C NMR spectrum of MAG derived from blood glucose of a fasted rat given the mixture with [U-13C3]glycerol, whereas B shows that of a fasted rat given the mixture with [U-13C3]lactate. Rats given [U-13C3]glycerol had large differences in the ratios of [1,2-13C2]/[2,3-13C2] and [5,6-13C2]/[4,5-13C2] in glucose. Although these differences were present in rats given [U-13C3]lactate, the effect was less substantial, demonstrating the advantage of [U-13C3]glycerol over [U-13C3]lactate in the estimation of hepatic PPP activity (C). Consequently, rats given [U-13C3]glycerol had more [1,2-13C2]glucose in blood produced by the hepatic PPP activity compared with rats given [U-13C3]lactate (D). D12, doublet from coupling of C1 with C2; D23, doublet from coupling of C2 with C3; Q, doublet of doublets, or quartet, arising from coupling of C2 with both C1 and C3 or from coupling of C5 with both C4 and C6; D45, doublet from coupling of C4 with C5; D56, doublet from coupling of C5 with C6; S, singlet (n = 5–7 in each group).

FIGURE 5.

Effects of dose and duration of [U-13C3]glycerol administration.A and B show 13C NMR spectra of MAG from blood glucose of a fasted (A) or fed (B) rat, and both rats received 100 mg/kg of [U-13C3]glycerol (50%), and blood was drawn at 60 min after the glycerol administration. In fasted rats, the ratio difference between [1,2-13C2]/[2,3-13C2] and [5,6-13C2]/[4,5-13C2] in glucose remained the same statistically over 180 min duration whether they received 50 or 100 mg/kg [U-13C3]glycerol (50%). However, fasted rats had higher ratio difference than fed animals when both groups received the same dose and duration of [U-13C3]glycerol administration (i.e. 100 mg/kg and 60 min; p = 0.004; C). The level of [1,2-13C2]glucose produced through the hepatic PPP (×2 due to 50% tracer) was higher in rats with 100 mg/kg [U-13C3]glycerol (50%) compared with rats with 50 mg/kg. Fasted rats had more [1,2-13C2]glucose produced by the PPP than fed animals (D) (n = 4–6 in each group).