On the inhibition of hepatic glycogenolysis by fructose. A 31P-NMR study in perfused rat liver using the fructose analogue 2,5-anhydro-D-mannitol
✍ Scribed by K. Bruynseels; N. Bergans; N. Gillis; F. van Dorpen; P. Van Hecke; W. Stalmans; F. Vanstapel
- Publisher
- John Wiley and Sons
- Year
- 1999
- Tongue
- English
- Weight
- 156 KB
- Volume
- 12
- Category
- Article
- ISSN
- 0952-3480
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✦ Synopsis
Inhibition of hormone-stimulated hepatic glycogenolysis by fructose (Fru) has been attributed to accumulation of the competitive inhibitor Fru1P 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-cAMP (100 mM) to clamp phosphorylase in its fully activated a form. We administered aHMol (3.8 mM), 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 aHMol1P 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 aHMol1P. Inhibition of glycogenolysis in excess of the drop in P i could be explained by continuing accumulation of aHMol1P. A subsequent block of mitochondrial ATP synthesis by KCN (1 mM) 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 aHMol1P into aHMol(1,6)P 2 . Residual inhibition of glycogenolysis was due to aHMol1P. Indeed, the subsequent withdrawal of aHMol caused a further gradual decrease in the proportion of aHMol1P (being converted into aHMol(1,6)P 2 , in the absence of de novo aHMol1P 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 aHMol1P 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