Nuclear magnetic resonance studies of carbohydrate metabolism and substrate cycling in Fasciola hepatica.
We have been interested in clarifying unique features of glycolytic metabolism in parasitic trematodes and in developing improved methods for monitoring the effects of pharmacologic agents that may alter functions of the pathway. In the present study metabolism of [1-13C]glucose by the common liver fluke, Fasciola hepatica, was studied both directly with 13C NMR and indirectly by observation of 13C-induced multiplet splitting of the 1H NMR resonances from the glycolytic end-products propionate and acetate. The extent of 13C enrichment of the end-products demonstrated that exogenous glucose was the predominant source of glycolytic substrate under the incubation conditions used. Specific enrichments of propionate and acetate in 13C were similar and enrichments at the acetate C-1 carboxyl and C-2 methyl were identical, demonstrating that acetate is generated preferentially from pyruvate formed by the malic enzyme reaction. End-product synthesized in substrate-free medium following incorporation of a small fraction of [1-13C]glucose into endogenous glycogen demonstrates that glucose equivalents from the most recently synthesized polymeric chains, which have a specific activity in 13C equal to that of the exogenous glucose, are preferentially used for glycogenolysis. Stimulation of flukes with 0.1 mM serotonin results in a reduction of the propionate/acetate 13C enrichment ratio consistent with functional "compartmentation" of glycogen pools having different structures and/or specific enrichment in 13C. Glucose equivalents were incorporated into glycogen in intact flukes with label at both the C-1 and C-6 positions during perfusion with [1-13C]glucose as a consequence of "substrate cycling" at the phosphofructokinase/fructosebisphosphatase enzyme couple. The observed glycogen C-6/C-1 labeling ratio of 0.42 and the net glycolytic flux of 11 mumol/g wet weight/hr imply a total forward flux of about 29 mumol/g wet weight/hr through phosphofructokinase with a reverse flux of about 17 mumol/g wet weight/hr through fructosebisphosphatase. Net glycolytic flux is therefore a poor estimate of the true flux through phosphofructokinase in this preparation.
D000085:Acetates / Q000378:metabolism; D019342:Acetic Acid; D000818:Animals; D050260:Carbohydrate Metabolism; D002247:Carbon Isotopes; D002417:Cattle; D005210:Fasciola hepatica / Q000378:metabolism*; D005635:Fructosediphosphates / Q000378:metabolism; D005636:Fructosephosphates / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D006003:Glycogen / Q000378:metabolism; D006019:Glycolysis; D009682:Magnetic Resonance Spectroscopy; D011422:Propionates / Q000378:metabolism; D011773:Pyruvates / Q000378:metabolism; D019289:Pyruvic Acid; D012701:Serotonin / Q000494:pharmacology
D000085:Acetates; D002247:Carbon Isotopes; D005635:Fructosediphosphates; D005636:Fructosephosphates; D011422:Propionates; D011773:Pyruvates; D012701:Serotonin; C027618:fructose-6-phosphate; D019289:Pyruvic Acid; D006003:Glycogen; D005947:Glucose; C029658:propionic acid; C029063:fructose-1,6-diphosphate; D019342:Acetic Acid