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Purpose: The positional analysis of hepatic glycogen enrichment from deuterated water ( 2 H 2 O) by 2 H NMR has been applied previously to resolve the contributions of glucose and fructose to glycogen synthesis in rodents fed a high sucrose diet. To further validate this method, this analysis was applied to mice fed with synthetic diets whose carbohydrate components consisted solely of either glucose or fructose., Methods: Eight glucose-fed and 12 fructose-fed mice were given 2 H 2 O followed by ad libitum feeding overnight. Mice were then euthanized, hepatic glycogen was isolated and derivatized to monoacetone glucose, and 2 H-enrichment of positions 2, 5, and 6 S were measured by 2 H NMR. From these data, the fraction of overnight glycogen appearance from the direct pathway and/or glycogen cycling and indirect pathway were estimated. Indirect pathway fractions were resolved into Krebs cycle and triose-phosphate sources-the latter including contributions from fructose metabolism., Results: After overnight feeding, the fraction of overnight glycogen appearance derived from direct pathway and/or glycogen cycling in glucose-fed-mice was 63 ± 1%. For the indirect pathway, Krebs cycle and triose-phosphate sources contributed 22 ± 1% and 15 ± 1%, respectively. For fructose-fed-mice, glycogen appearance was dominated by triose-phosphate sources (60 ± 2%) with lesser contributions from Krebs cycle (14 ± 1%) and direct and/or glycogen cycling (26 ± 2%)., Conclusions: 2 H NMR analysis of hepatic glycogen 2 H enrichment from 2 H 2 O provides realistic profiles of dietary glucose and fructose contributions to hepatic glycogen synthesis in mice fed with diets containing 1 or the other sugar as the sole carbohydrate source., (© 2018 International Society for Magnetic Resonance in Medicine.)

Farmed seabass have higher adiposity than their wild counterparts and this is often attributed to carbohydrate (CHO) feeding. Whether this reflects a reduction in fat oxidation, increased de novo lipogenesis (DNL), or both, is not known. To study the effects of high CHO diets on hepatic TG biosynthesis, hepatic TG deuterium ((2)H) enrichment was determined following 6 days in (2)H-enriched tank water for fish fed with a no-CHO control diet (CTRL), and diets with digestible starch (DS) and raw starch (RS). Hepatic fractional synthetic rates (FSRs, percent per day(-1)) were calculated for hepatic TG-glyceryl and FA moieties through (2)H NMR analysis. Glyceryl FSRs exceeded FA FSRs in all cases, indicating active cycling. DS fish did not show increased lipogenic potential compared to CTRL. RS fish had lower glyceryl FSRs compared with the other diets and negligible levels of FA FSRs despite similar hepatic TG levels to CTRL. DS-fed fish showed higher activity for enzymes that can provide NADPH for lipogenesis, relative to CTRL in the case of glucose-6-phosphate dehydrogenase (G6PDH) and relative to RS for both G6PDH and 6-phosphogluconate dehydrogenase. This approach indicated that elevated hepatic adiposity from DS feeding was not attributable to increased DNL., (Copyright © 2016 by the American Society for Biochemistry and Molecular Biology, Inc.)

In the present study, the effects of partial substitution of dietary protein by digestible starch on endogenous glucose production were evaluated in European seabass (Dicentrarchus labrax). The fractional contribution of dietary carbohydrates v. gluconeogenesis to blood glucose appearance and hepatic glycogen synthesis was quantified in two groups of seabass fed with a diet containing 30% digestible starch (DS) or without a carbohydrate supplement as the control (CTRL). Measurements were performed by transferring the fish to a tank containing water enriched with 5% (2)H2O over the last six feeding days, and quantifying the incorporation of (2)H into blood glucose and hepatic glycogen by (2)H NMR. For CTRL fish, gluconeogenesis accounted for the majority of circulating glucose while for the DS fish, this contribution was significantly lower (CTRL 85 (SEM 4) % v. DS 54 (SEM 2) %; P < 0.001). Hepatic glycogen synthesis via gluconeogenesis (indirect pathway) was also significantly reduced in the DS fish, in both relative (CTRL 100 (SEM 1) % v. DS 72 (SEM 1) %; P < 0.001) and absolute terms (CTRL 28 (SEM 1) v. DS 17 (sem 1) μmol/kg per h; P < 0.001). A major fraction of the dietary carbohydrates that contributed to blood glucose appearance (33 (sem 1) % of the total 47 (SEM 2) %) had undergone exchange with hepatic glucose 6-phosphate. This indicated the simultaneous activity of hepatic glucokinase and glucose 6-phosphatase. In conclusion, supplementation of digestible starch resulted in a significant reduction of gluconeogenic contributions to systemic glucose appearance and hepatic glycogen synthesis.

The stimulation of hepatic glycogenesis is a ubiquitous response to a glucose challenge and quantifying its contribution to glucose uptake informs its role in restoring euglycemia. Glycogenesis can be quantified with labeled water provided that exchange of glucose-6-phosphate hydrogen 2 (G6P-H2) and body water via glucose-6-phosphate isomerase, and exchange of positions 4, 5 and 6 hydrogens (G6P-H456) via transaldolase, are known. These exchanges were quantified in 24-h fasted rats (Rattus norvegicus; n=6) and 21-day fasted seabass (Dicentrarchus labrax; n=8) by administration of a glucose load (2000mg·kg(-1)) enriched with [U-(2)H7]glucose and by quantifying hepatic glycogen (2)H-enrichments after 2h (rats) and 48h (seabass). Direct pathway contributions of the glucose load to glycogenesis were also estimated. G6P-H2 and body water exchange was 61±1% for rat and 47±3% for seabass. Transaldolase-mediated exchange of G6P-H456 was 5±1% for rat and 10±1% for seabass. Conversion of the glucose load to hepatic glycogen was significant in seabass (249±54mg·kg(-1)) but negligible in rats (12±1mg·kg(-1)). Preload plasma glucose levels were similar for seabass and rats (3.3±0.7 and 4.4±0.1mmol·L(-1), respectively) but post-load plasma glucose was significantly higher in seabass compared to rats (14.6±1.8 versus 5.8±0.3mmol·L(-1), p<0.01). In conclusion, G6P-H2 and body water exchange is incomplete for both species and has to be accounted for in estimating hepatic glycogen synthesis and direct pathway activities with labeled water tracers. Transaldolase-mediated exchange is insignificant. Hepatic direct pathway glycogenesis plays a prominent role in seabass glucose load disposal, but a negligible role in the rat., (© 2013.)

Hepatic glycogen synthesis fluxes from direct and indirect pathways were quantified in seabass by postmortem (2)H NMR analysis of plasma water (PW) and glycogen glucosyl (2)H enrichments from (2)H-enriched seawater. Eighteen fish (28.0 ± 1.7 cm and 218.0 ± 43.0 g) were divided into three groups of 6 and studied over 24 days with transfer to 5% (2)H-seawater after day 21. Over this period, one group was fed daily with fishmeal, a second group was fasted, and a third group was fasted for 21 days followed by 3 days refeeding. Glycogen turnover and sources were determined from the ratio of glucosyl position 5 enrichment to that of plasma water (H5/PW). Glycogen levels of fed fish were significantly higher than fasted (665.4 ± 345.2 μmol.g(-1) liver versus 77.2 ± 59.5 μmol.g(-1) liver, P<0.05) while refed fish had comparable levels to fed (584.6 ± 140.4 μmol.g(-1) liver). Glycogen enrichment of fed fish was undetectable indicating negligible turnover over 3 days. For fasted fish, H5/PW was ~50% indicating that half of the glycogen had turned over via indirect pathway flux. For refed fish, H5/PW was ~100% indicating that the indirect pathway accounted for all net glycogen synthesis. Direct pathway conversion of dietary carbohydrate to glycogen was not detected in any of the groups., (Copyright © 2012 Elsevier Inc. All rights reserved.)