Quantification of the glycogen 13C-1 NMR signal during glycogen synthesis in perfused rat liver

We studied glycogen synthesis from glucose in perfused livers of fed (n = 4) and 24 h starved (n = 7) rats. Glycogenolysis was inhibited by BAY R3401 (150 muM) and proglycosyn (100 muM). After 60 min, we replaced 99% C-13-1 glucose by natural abundance glucose. This pulse-chase design allowed us to recognize residual ongoing futile glycogen turnover from the release of initially deposited C-13-label, into the C-13-free chase medium. Net residual turnover was less than 2 +/- 0.7% and 0.6 +/- 0.2% of l-C-13 glycogen deposition rates of 0.31 +/- 0.04 and 0.99 +/- 0.04 mumol glucose g(-1) min(-1), in starved and fed livers, respectively. The l-C-13 glycogen signal was monitored throughout the experiment with proton-decoupled C-13 NMR spectroscopy and analyzed in the time domain using AMARES. We noticed progressive line-broadening in any single experiment in the chase phase. One or a sum of two to three overlapping Lorentzians, with different exponential damping factors, were fitted to the signal. When the S/N was better than 40, the fit always delivered a small and a broad component. In the chase phase, the fit with a single Lorentzian resulted in a decline of glycogen signal by about 15 +/- 4 and 12 +/- 2% in starved and fed rats, respectively. This apparent decline in l-C-13 glycogen signal could not be accounted for by the appearance of equivalent amounts of C-13-labeled metabolites in the perfusate. The fit with a sum of two Lorentzians resulted in a decline of glycogen signal intensity of 7 +/- 5 and 5 +/- 3% in starved and fed rats, respectively, which reduced the apparent turnover to 8 +/- 9% and 6 +/- 4%, respectively. Quantification of the growing C-13-1 glycogen signal requires a model function that accommodates changes in line shape throughout the period under study. Copyright (C) 2003 John Wiley Sons, Ltd.