Sheila M. Cohen

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.

Studies of density dependence of 129Xe chemical shift in xenon gas at room temperature have shown that while the chemical shielding does have quadratic and cubic dependence on density over densities up to 250 amagat, σ(ρ, T) = σ0 + σ1(T)ρ + σ2(T)ρ2 + σ3(T)ρ3, the curve is essentially linear up to about 100 amagat. We have now obtained σ1(T), the linear density coefficient of chemical shielding, for pure xenon over the temperature range 240–440 °K. The experimental values of σ1(T) can be fitted by a fourth degree polynomial: σ1(τ) = 0.536 − 0.135 × 10−2τ + 0.132 × 10−4 τ2 − 0.598 × 10−7τ3 + 0.663 × 10−10τ4 (ppm/amagat), where τ = T − 300 °K. Comparison is made with σ1(T) for other nuclei and with σ1(T) predicted by various theoretical models.

Since the pioneer paper of Moon & Richards in 1973 recording the 31p NMR spectrum of red blood cells (63), application of 31 P NMR to biologi­ cal systems has become very popular. Studies have ranged from the simple observation of anaerobic metabolism to the elegant combination of physiol­ ogy and spectroscopy demonstrated by Dawson et al (27) on frog muscle. Some reviews appeared on this topic (12, 30, 37). The low number of metabolites that contain phosphorus at levels that can be seen in the spectra (>0.5 mM in 1 5 min of observation time) differentiates phosphorus NMR from carbon and proton NMR, where the vast number of carbonand proton-containing compounds, except under special circumstances, yield complicated spectra that are not easily interpretable (26). The 31 P spectra obtained in all tissues examined are relatively uncomplicated and hence readily interpretable. With the demon­ stration of the feasibility of studying intact tissue, a door has been opened that allows the examination of many critical issues in biochemistry and physiology from a totally new angle. We document how this has ranged from the identification of previously undetected phosphates to tests of hypotheses of bioenergetics. 1

A method is proposed for measuring on the bench the NMR signal-to-noise ratio of rf probes, (over the range 1-100 MHz) and also the power deposited in patients during the imaging experiment. The technique is based on the principle of reciprocity, in that a direct relationship exists between the magnetic field generated (upon transmission) by a matched probe coil and the signal-to-noise ratio delivered by the same coil when used as a receiver. The construction and use of a calibrated sense coil for measuring the field is described, and the precautions and theory necessary for accurate measurement and understanding are outlined. Finally, the method is verified by comparison with a direct spectral measure of sensitivity obtained from a small doped water sample placed in NMR imaging equipment.

Glucagon maintains glucose homeostasis during the fasting state by promoting hepatic gluconeogenesis and glycogenolysis. Hyperglucagonemia and/or an elevated glucagon-to-insulin ratio have been reported in diabetic patients and animals. Antagonizing the glucagon receptor is expected to result in reduced hepatic glucose overproduction, leading to overall glycemic control. Here we report the discovery and characterization of compound 1 (Cpd 1), a compound that inhibits binding of 125I-labeled glucagon to the human glucagon receptor with a half-maximal inhibitory concentration value of 181 +/- 10 nmol/l. In CHO cells overexpressing the human glucagon receptor, Cpd 1 increased the half-maximal effect for glucagon stimulation of adenylyl cyclase with a KDB of 81 +/- 11 nmol/l. In addition, Cpd 1 blocked glucagon-mediated glycogenolysis in primary human hepatocytes. In contrast, a structurally related analog (Cpd 2) was not effective in blocking glucagon-mediated biological effects. Real-time measurement of glycogen synthesis and breakdown in perfused mouse liver showed that Cpd 1 is capable of blocking glucagon-induced glycogenolysis in a dosage-dependent manner. Finally, when dosed in humanized mice, Cpd 1 blocked the rise of glucose levels observed after intraperitoneal administration of exogenous glucagon. Taken together, these data suggest that Cpd 1 is a potent glucagon receptor antagonist that has the capability to block the effects of glucagon in vivo.