Estimating pentose phosphate pathway activity from the analysis of hepatic glycogen 13 C-isotopomers derived from [U-13 C]fructose and [U-13 C]glucose.

Abstract

The pentose phosphate pathway (PPP) is an important component of hepatic intermediary metabolism. Jin et al developed an elegant 13 C-NMR method for measuring hepatic PPP flux by quantifying the distribution of glucose 13 C-isotopomers formed from [U-13 C]glycerol. We demonstrate that this approach can be extended to exogenous [U-13 C]fructose and [U-13 C]glucose precursors by 13 C-NMR analysis of glycogen. Twelve male C57BL/6 mice fed standard chow were provided a 55/45 mixture of fructose and glucose at 30% w/v in the drinking water for 18 wk. On the evening before sacrifice, the fructose component was enriched with 20% [U-13 C]fructose for 6 mice, while the glucose component was enriched with 20% [U-13 C]glucose for the remaining 6 mice. Mice were allowed to feed and drink naturally overnight, and then, euthanized. Livers were freeze-clamped and glycogen was extracted and derivatized for 13 C NMR spectroscopy. Flux of each sugar into the PPP relative to its incorporation into glycogen was quantified from selected 13 C glycogen isotopomer ratios. Both [U-13 C]fructose and [U-13 C]glucose precursors yielded glycogen 13 C-isotopomer distributions that were characteristic of PPP activity. The fraction of [U-13 C]glucose utilized by the PPP relative to its conversion to glycogen via the direct pathway was 14 ± 1%, while that from [U-13 C]fructose relative to its conversion to glycogen via the indirect pathway was significantly lower (10 ± 1%, P = .00032). Hepatic PPP fluxes from both [U-13 C]glucose and [U-13 C]fructose precursors were assessed by 13 C NMR analysis of glycogen 13 C-isotopomers. Glucose-6-phosphate generated via glucokinase and the direct pathway is preferentially utilized by the PPP.