13C and 1H nuclear magnetic resonance study of glycogen futile cycling in strains of the genus Fibrobacter

Abstract

We investigated the carbon metabolism of three strains of Fibrobacter succinogenes and one strain of Fibrobacter intestinalis. The four strains produced the same amounts of the metabolites succinate, acetate, and formate in approximately the same ratio (3.7/1/0.3). The four strains similarly stored glycogen during all growth phases, and the glycogen-to-protein ratio was close to 0.6 during the exponential growth phase. 13C nuclear magnetic resonance (NMR) analysis of [1-13C]glucose utilization by resting cells of the four strains revealed a reversal of glycolysis at the triose phosphate level and the same metabolic pathways. Glycogen futile cycling was demonstrated by 13C NMR by following the simultaneous metabolism of labeled [13C]glycogen and exogenous unlabeled glucose. The isotopic dilutions of the CH2 of succinate and the CH3 of acetate when the resting cells were metabolizing [1-13C]glucose and unlabeled glycogen were precisely quantified by using 13C-filtered spin-echo difference 1H NMR spectroscopy. The measured isotopic dilutions were not the same for succinate and acetate; in the case of succinate, the dilutions reflected only the contribution of glycogen futile cycling, while in the case of acetate, another mechanism was also involved. Results obtained in complementary experiments are consistent with reversal of the succinate synthesis pathway. Our results indicated that for all of the strains, from 12 to 16% of the glucose entering the metabolic pathway originated from prestored glycogen. Although genetically diverse, the four Fibrobacter strains studied had very similar carbon metabolism characteristics.

FIG. 1

¹³C NMR spectra of in vivo [1-¹³C]glucose utilization by resting cells of Fibrobacter species. [1-¹³C]glucose (32 mM) was added to a cell suspension (5 mg of protein · ml⁻¹) in a 10-mm tube, and proton-decoupled ¹³C NMR spectra were obtained every 4.5 min; the spectra shown were recorded 22.5 min after glucose was added. AC, acetate; GLC, glucose; GLY, glycogen; MAL, malate; SUC, succinate.

FIG. 2

Time-dependent changes in signal integrals of labeled metabolites during [1-¹³C]glucose or prestored [1-¹³C]glycogen and [6-¹³C]glycogen utilization by resting cells of F. intestinalis NR9. (A) Resting cells of F. intestinalis (5 mg of protein · ml⁻¹) were incubated with 32 mM [1-¹³C]glucose, and metabolite production was monitored during glucose utilization and 25 min after exogenous carbon sources were exhausted. (B) Resting cells of F. intestinalis (5 mg of protein · ml⁻¹) were first incubated with 32 mM [1-¹³C]glucose for 22.5 min, washed, and incubated with 32 mM unlabeled glucose. (C) Resting cells of F. intestinalis (5 mg of protein · ml⁻¹) were first incubated with 32 mM unlabeled glucose for 20 min, washed, and incubated with 32 mM [1-¹³C]glucose. Symbols: □, total [1-¹³C]glucose; ▴, total [1-¹³C]glucose 6-phosphate; •, [1-¹³C]glycogen; ○, [6-¹³C]glycogen; ▪, total [1-¹³C]glycogen and [6-¹³C]glycogen; ▵, [2-¹³C]succinate.

FIG. 3

¹³C NMR spectra for in vivo prestored [1-¹³C]glycogen and [6-¹³C]glycogen utilization by resting cells of Fibrobacter species. Resting Fibrobacter cells (5 mg of protein · ml⁻¹) were first incubated with 32 mM [1-¹³C]glucose for 22.5 min, washed, and incubated with 32 mM unlabeled glucose. The proton-decoupled ¹³C NMR spectra shown were recorded 27 min after unlabeled glucose was added. GLY, glycogen; G6P, glucose 6-phosphate; SUC, succinate.

FIG. 4

Separation of ¹²C and ¹³C subspectra in ¹H NMR spectra from strain HM2. The spectra were acquired with an X-filtered spin-echo pulse sequence as described in Materials and Methods. Spectrum A represents the output of an experiment with no 180° (¹³C) pulse in the middle of the spin-echo period and is similar to a standard ¹H NMR spectrum. Subspectra B and C were obtained after subtraction (subspectrum B) or addition (subspectrum C) of the two original spectra acquired with the spin-echo pulse sequence (with and without central inversion of ¹³C spin). Subspectrum C is a pure ¹³C isotopomer subspectrum of a standard ¹H spectrum.

FIG. 5

Proton-decoupled ¹³C NMR spectra obtained with extracts of F. succinogenes S85 incubated with [1-¹³C]glucose (spectrum A), [2-¹³C]glucose (spectrum B), or [¹²C]glucose (spectrum C) by using an inverse-gated sequence.

FIG. 6

Expected enrichment of succinate and acetate produced from 100% [1-¹³C]glucose. The asterisk indicates the ¹³C atom.