Simultaneous 13C and 31P NMR studies of perfused rat liver. Effects of insulin and glucagon and a 13C NMR assay of free Mg2+.

Abstract

Metabolism of [2-13C]pyruvate, [1,2-13C]ethanol, and NH4+ in the presence and absence of 7 nM insulin has been followed at 35 degrees C by alternate scan 13C and 31P NMR at 90.5 and 145.8 MHz, respectively, in isolated perfused liver from 16-h fasted rats. With this technique, 31P and 13C NMR spectra are recorded simultaneously so that both phosphate metabolites and 13C-labeled metabolites could be followed, noninvasively, in perfused liver to give a comprehensive view of the response to a variety of stimuli. 13C-labeled glycogen increased synchronously, at a rate of 17 mumol of glucose units/g of liver/h, with the synthesis of 13C-labeled glucose, which also proceeded at a rate of 17 mumol/g of liver/h; glycogenesis was essentially a gluconeogenic process under these conditions and was not affected by the presence of insulin. From the position of the 13C-labeled citrate peak observed in liver, the measurement of Kd for the citrate-Mg complex under our conditions, and the expression relating these quantities to the concentration of free Mg2+, the intracellular level of free Mg2+ is estimated to be 0.46 +/- 0.05 mM in perfused rat liver. After subsequent administration of glucagon, a rapid decrease in glycogen and citrate was seen by 13C NMR and a 44% increase in glycero-3-phosphocholine was seen by 31P NMR; increase in glycero-3-phosphocholine is consistent with stimulation of liver phospholipase activity by glucagon. The co-administration of two different 13C-labeled substrates introduced multiplet structure arising from spin-spin interaction between labeled adjacent carbons into the peaks of several key metabolites. 13C enrichments at specific carbons of citrate, glutamate, glutamine, beta-hydroxybutyrate, and glucose and the distribution of intensity within the multiplets of specific carbons were measured in spectra of perfusates and extracts of the freeze-clamped livers. Within the context of a first order model for fluxes into the Krebs cycle and into glucose, analytical expressions were written that describe the intensity distributions within the several multiplets. In this way, a set of simultaneous equations was generated and solved in general form; when the measured intensity ratios are substituted into these expressions, relative fluxes under the conditions of the experiment can be estimated. Because a redundancy of information is available, checks on self-consistency are built into the estimated fluxes.