Enrichment of hepatic glycogen and plasma glucose from H₂(18)O informs gluconeogenic and indirect pathway fluxes in naturally feeding mice

Deuterated water ((2)H(2)O) is a widely used tracer of carbohydrate biosynthesis in both preclinical and clinical settings, but the significant kinetic isotope effects (KIE) of (2)H can distort metabolic information and mediate toxicity. (18)O‐water (H(2) (18)O) has no significant KIE and is incorporated into specific carbohydrate oxygens via well‐defined mechanisms, but to date it has not been evaluated in any animal model. Mice were given H(2) (18)O during overnight feeding and (18)O‐enrichments of liver glycogen, triglyceride glycerol (TG), and blood glucose were quantified by (13)C NMR and mass spectrometry (MS). Enrichment of oxygens 5 and 6 relative to body water informed indirect pathway contributions from the Krebs cycle and triose phosphate sources. Compared with mice fed normal chow (NC), mice whose NC was supplemented with a fructose/glucose mix (i.e., a high sugar [HS] diet) had significantly higher indirect pathway contributions from triose phosphate sources, consistent with fructose glycogenesis. Blood glucose and liver TG (18)O‐enrichments were quantified by MS. Blood glucose (18)O‐enrichment was significantly higher for HS versus NC mice and was consistent with gluconeogenic fructose metabolism. TG (18)O‐enrichment was extensive for both NC and HS mice, indicating a high turnover of liver triglyceride, independent of diet. Thus H(2) (18)O informs hepatic carbohydrate biosynthesis in similar detail to (2)H(2)O but without KIE‐associated risks.