Limitations of the Mass Isotopomer Distribution Analysis of Glucose to Study Gluconeogenesis

Abstract

Mass isotopomer distribution analysis allows studying the synthesis of polymeric biomolecules from 15N, 13C-, or 2H-labeled monomeric units in the presence of unlabeled polymer. The mass isotopomer distribution of the polymer allows calculation of (i) the enrichment of the monomer and (ii) the dilution of the newly synthesized polymer by unlabeled polymer. We tested the conditions of validity of mass isotopomer distribution analysis of glucose labeled from [U-13C3]lactate, [U-13C3]glycerol, and [2-13C]glycerol to calculate the fraction of glucose production derived from gluconeogenesis. Experiments were conducted in perfused rat livers, live rats, and live monkeys. In all cases, [13C]glycerol yielded labeling patterns of glucose that are incompatible with glucose being formed from a single pool of triose phosphates of constant enrichment. We show evidence that variations in the enrichment of triose phosphates result from (i) the large fractional decrease in physiological glycerol concentration in a single pass through the liver and (ii) the release of unlabeled glycerol by the liver, presumably via lipase activity. This zonation of glycerol metabolism in liver results in the calculation of artifactually low contributions of gluconeogenesis to glucose production when the latter is labeled from [13C]glycerol. In contrast, [U-13C3]lactate appears to be a suitable tracer for mass isotopomer distribution analysis of gluconeogenesis in vivo, but not in the perfused liver. In other perfusion experiments with [2H5]glycerol, we showed that the rat liver releases glycerol molecules containing one to four 2H atoms. This indicates the operation of a substrate cycle between extracellular glycerol and liver triose phosphates, where 2H is lost in the reversible reactions catalyzed by alpha-glycerophosphate dehydrogenase, triose-phosphate isomerase, and glycolytic enzymes. This substrate cycle presumably involves alpha-glycerophosphate hydrolysis.

Hepatic gluconeogenic fluxes and glycogen turnover during fasting in humans. A stable isotope study.

“…The latter form of glycogen cycling represented 20-40% of UDP-glucose flux; this component of glycogen turnover is indeed ordered (last in/ first out) under nonaccumulating conditions. An important implication of these results is that the correct value for isotopically measured f Glc need not be 100%, and indeed is not 100%, after a prolonged fast, contrary to the common assumption (2,41). In fact, certain techniques used previously for measuring f Glc that were reported as being close to 100% in fasted humans (e.g., the [2- (16,26,42) and mice (our unpublished observations), and in perfused livers from fasted rats (42).…”

Physiologic and Pharmacologic Factors Influencing GlyceroneogenicContribution to Triacylglyceride Glycerol Measured by Mass IsotopomerDistribution Analysis

“…Second, glycolytic cycling of glucose to the level of pyruvate or oxaloacetate then back to ␣-GP can contribute to TG (7,36). Cycling of this type converts glyc 3.5 to glyc 5 , and thus can complicate estimates of the proportion of TG-glycerol derived from glycolysis.…”

Quantitation of Gluconeogenesis by 2H Nuclear Magnetic Resonance Analysis of Plasma Glucose Following Ingestion of 2H2O

“…Therefore, enrichment of 2 H 2 O across the liver is uniform and the gluconeogenic flux estimate is representative of the whole liver. In comparison, carbon tracers of gluconeogenesis are not always evenly distributed across the liver and therefore may not provide representative estimates of hepatic gluconeogenesis (20,21). However, carbon tracers are essential for measuring metabolic fluxes at the Krebs cycle level (22)(23)(24)(25), the principal source of both carbons and energy for gluconeogenesis.…”

“…Since glycerol normally contributes less than 15% of the total gluconeogenic carbons (26,27), underrepresentation of total gluconeogenesis should be small given the extent of G3P-DHAP randomization. Cycling between glycerol and DHAP (20) provides an additional opportunity for enriching glucose H3 from glycerol. In comparison, the stoichiometry between glucose H5 enrichment and gluconeogenesis is preserved regardless of the source of gluconeogenic carbons and the extent of triose phosphate randomization (2).…”