Submitted to: American Journal of Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/27/2000
Publication Date: 2/1/2001
Interpretive Summary: Measurement of the rate of a metabolic pathway is central and fundamental to the proper assessment of the contribution of a given pathway to the health of a tissue. Glycolysis is the primary metabolic pathway for the consumption of glucose. In order to measure the rate of this pathway, researchers often give a trace amount of radioactive glucose ([5-3H] glucose) and measure the rate of radioactive water ([3H]H20) produced in the tissue. In the present study, we used this method as well as two other methods for measuring the rate of glycolysis in the perfused rat heart. We found that the method using radioactive glucose overestimated the true rate of glycolysis, and we suggest that the reason is the presence of transaldolase activity (which also produces radioactive water from the radioactive glucose). The results of this study will have impact on all other studies of heart metabolism in which the role of glycolysis was assessed.
Technical Abstract: We studied 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 muU/ml insulin, 0.5 5mM lactate, 0.05 mM pyruvate and 0.4 mM oleate/3% albumin. Results: The steady-state C1/C6 ratio (i.e., the ratio from [1-14C] glucose/[6-14C] glucose) of metabolites released by the heart, an index of oxidative PPP, was not different from one (1.06 +/- 0.19 for 14CO2 and 1.00 +/- 0.01 for 14C-lactate + 14C-pyruvate, mean +/- S.E., n=8). Hearts exhibited contractile, metabolic, and 14C-isotopic steady-state for glucose oxidation (14CO2 production). Net glucolytic 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) were not different from each other, and were less than one (0.36 +/- 0.01 and 0.43 +/- 0.05, respectively, n=8, P<0.05 vs. 1). Conclusions: 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 true glycolytic flux in rat heart.