[5-³H]glucose overestimates glycolytic flux in isolated working rat heart: role of the pentose phosphate pathway

Abstract

We set out to study the pentose phosphate pathway (PPP) in isolated rat hearts perfused with [5-3H]glucose and [1-14C]glucose or [6-14C]glucose (crossover study with 1- then 6- or 6- then 1-14C-labeled glucose). To model a physiological state, hearts were perfused under working conditions with Krebs-Henseleit buffer containing 5 mM glucose, 40 microU/ml insulin, 0.5 mM lactate, 0.05 mM pyruvate, and 0.4 mM oleate/3% albumin. The steady-state C1/C6 ratio (i.e., the ratio from [1-14C]glucose to [6-14C]glucose) of metabolites released by the heart, an index of oxidative PPP, was not different from 1 (1.06 +/- 0.19 for 14CO2, and 1.00 +/- 0.01 for [14C]lactate + [14C]pyruvate, mean +/- SE, n = 8). Hearts exhibited contractile, metabolic, and 14C-isotopic steady state for glucose oxidation (14CO2 production). Net glycolytic flux (net release of lactate + pyruvate) and efflux of [14C]lactate + [14C]pyruvate were the same and also exhibited steady state. In contrast, flux based on 3H2O production from [5-3H]glucose increased progressively, reaching 260% of the other measures of glycolysis after 30 min. The 3H/14C ratio of glycogen (relative to extracellular glucose) and sugar phosphates (representing the glycogen precursor pool of hexose phosphates) was not different from each other and was <1 (0.36 +/- 0.01 and 0.43 +/- 0.05 respectively, n = 8, P < 0.05 vs. 1). We conclude that both transaldolase and the L-type PPP permit hexose detritiation in the absence of net glycolytic flux by allowing interconversion of glycolytic hexose and triose phosphates. Thus apparent glycolytic flux obtained by 3H2O production from [5-3H]glucose overestimates the true glycolytic flux in rat heart.

Underestimation of D-glucose utilisation as judged from the conversion of D-[3-3H]glucose to 3HOH

“…Both phenomena could conceivably be attributed to the accumulation of glycogen in the beta cells of the hyperglycaemic GK rats and, hence, to the stimulation of glycogenolysis, with a subsequent increase in glycolytic flux, in the islets of GK rats exposed to low concentrations of D-glucose [23]. It has been proposed that, in the isolated working heart, 3 HOH production from D-[5-3 H]glucose could overestimate glycolytic flux, possibly through generation of 3 HOH in the L-type pentose phosphate pathway and/or through transaldolase exchange [26]. In this respect, it should also be kept in mind that the 3 H from D-[3-3 H]glucose is lost as NADP 3 H in the oxidative portion of the pentose phosphate pathway.…”

Adaptation and Maladaptation of the Heart in Diabetes: Part II

“…56,57 Appreciable flux through the non-oxidative PPP occurs in the heart. 76 In addition, pressure overload induces glucose 6-phosphate dehydrogenase (G6PDH), the enzyme catalyzing the flux generating step in the oxidative PPP. 77 It is therefore reasonable to assume that xylulose 5-phosphate levels increase in the hypertrophied heart due to the combined increase in glucose flux into the cardiomyocyte and increased G6PDH activity.…”

(R)-α-Lipoic acid reverses the age-related loss in GSH redox status in post-mitotic tissues: evidence for increased cysteine requirement for GSH synthesis

“…To initially characterize potential factors involved, the age-dependent changes in activities of GR and Grx were examined and reveal no significant alterations in their activities, indicating that aging may compromise cellular capacity to maintain pyridine nucleotide redox states and/or to export GSSG. NADPH production in tissues can arise from multiple pathways, including the pentose phosphate pathway (PPP), NADP + -dependent isocitrate dehydrogenase (ICDH), and transhydrogenation reactions [33][34][35]. Among these pathways, ICDH has been previously identified as the principal generator of NADPH in the heart, while the primary modes of NADPH production in the brain have not been fully defined [33,34].…”