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On the inhibition of hepatic glycogenolysis by fructose. A 31 P‐NMR study in perfused rat liver using the fructose analogue 2,5‐anhydro‐ D ‐mannitol
Author(s) -
Bruynseels K.,
Bergans N.,
Gillis N.,
van Dorpen F.,
Van Hecke P.,
Stalmans W.,
Vanstapel F.
Publication year - 1999
Publication title -
nmr in biomedicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.278
H-Index - 114
eISSN - 1099-1492
pISSN - 0952-3480
DOI - 10.1002/(sici)1099-1492(199905)12:3<145::aid-nbm559>3.0.co;2-1
Subject(s) - glycogenolysis , gluconeogenesis , chemistry , phosphomonoesters , fructose , endocrinology , substrate (aquarium) , medicine , pi , glycogen phosphorylase , biochemistry , glycogen , metabolism , phosphate , biology , ecology , inorganic phosphate
Inhibition of hormone‐stimulated hepatic glycogenolysis by fructose (Fru) has been attributed to accumulation of the competitive inhibitor Fru1 P and/or to the associated depletion of the substrate phosphate (P i ). To evaluate the relative importance of either factor, we used the Fru analogue 2,5‐anhydro‐ D ‐mannitol (aHMol). This analogue is avidly phosphorylated, traps P i , and inhibits hormone‐stimulated glycogenolysis, but it is not a gluconeogenic substrate, and hence does not confound glycogenolytic glucose production. Livers were continuously perfused with dibutyryl‐ c AMP (100 µ M ) to clamp phosphorylase in its fully activated a form. We administered aHMol (3.8 m M ), and studied changes in glycogenolysis (glucose, lactate and pyruvate output) and in cytosolic P i and phosphomonoester (PME), using in situ 31 P‐NMR spectroscopy ( n = 4). Lobes of seven livers perfused outside the magnet were extracted for evaluation, by high‐resolution 31 P‐NMR, of the evolution of aHMol1 P and of aHMol(1,6) P 2 . After addition of aHMol, both glycogenolysis and the NMR P i signal dropped precipitously, while the PME signal rose continuously and was almost entirely composed of aHMol1 P . Inhibition of glycogenolysis in excess of the drop in P i could be explained by continuing accumulation of aHMol1 P . A subsequent block of mitochondrial ATP synthesis by KCN (1 m M ) caused a rapid increase of P i . Despite recovery of P i to values exceeding control levels, glycogenolysis only recovered partially, attesting to the P i ‐dependence of glycogenolysis, but also to inhibition by aHMol phosphorylation products. However, KCN resulted in conversion of the major part of aHMol1 P into aHMol(1,6) P 2 . Residual inhibition of glycogenolysis was due to aHMol1 P . Indeed, the subsequent withdrawal of aHMol caused a further gradual decrease in the proportion of aHMol1 P (being converted into aHMol(1,6) P 2 , in the absence of de novo aHMol1 P synthesis), and this resulted in a gradual de‐inhibition of glycogenolysis, in the absence of marked changes in P i . Glycogenolytic rates were consistently predicted by a model assuming non‐saturated P i kinetics and competition by aHMol1 P exclusively: In conclusion, limited P i availability and the presence of competitive inhibitors are decisive factors in the control of the in situ catalytic potential of phosphorylase a . Copyright © 1999 John Wiley & Sons, Ltd.