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 microM) and proglycosyn (100 microM). After 60 min, we replaced 99% (13)C-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 (13)C-label, into the (13)C-free chase medium. Net residual turnover was less than 2 +/- 0.7% and 0.6 +/- 0.2% of 1-(13)C glycogen deposition rates of 0.31 +/- 0.04 and 0.99 +/- 0.04 micromol glucose g(-1) min(-1), in starved and fed livers, respectively. The 1-(13)C glycogen signal was monitored throughout the experiment with proton-decoupled (13)C 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 1-(13)C glycogen signal could not be accounted for by the appearance of equivalent amounts of (13)C-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 (13)C-1 glycogen signal requires a model function that accommodates changes in line shape throughout the period under study.
Bergans
N
N
Biomedische NMR Eenheid, Afdeling Röntgendiagnose, Katholieke Universiteit Leuven, Leuven, Belgium.
Dresselaers
T
T
Vanhamme
L
L
Van Hecke
P
P
Van Huffel
S
S
Vanstapel
F
F
D000465:Algorithms; D000818:Animals; D002247:Carbon Isotopes; D004095:Dihydropyridines / Q000494:pharmacology; D005508:Food Deprivation / Q000502:physiology; D005663:Furans / Q000494:pharmacology; D008099:Liver / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D008112:Liver Glycogen / Q000032:analysis* / Q000096:biosynthesis* / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008297:Male; D008954:Models, Biological; D010477:Perfusion; D051381:Rats; D017208:Rats, Wistar; D012815:Signal Processing, Computer-Assisted
C413462:BAY R3401; D002247:Carbon Isotopes; D004095:Dihydropyridines; D005663:Furans; D008112:Liver Glycogen